Solid- and solution-phase synthesis of heparin and other glycosaminoglycans

ABSTRACT

Described is a modular, general synthetic strategy for the preparation in solution and on a solid support of heparin, heparin-like glycosaminoglycans, glycosaminoglycans and non-natural analogs of each of them. Additionally, the modular strategy provides the basis for the preparation of combinatorial libraries and parallel libraries of defined glycosaminoglycan oligosaccharides. The defined glycosaminoglycan structures may be used in high-throughput screening experiments to identify carbohydrate sequences that regulate a host of recognition and signal-transduction processes. The determination of specific sequences involved in receptor binding holds great promise for the development of molecular tools which will allow modulation of processes underlying viral entry, angiogenesis, kidney diseases and diseases of the central nervous system. Notably, the present invention enables the automated synthesis of glycosaminoglycans in much the same fashion that peptides and oligonucleotides are currently assembled.

RELATED APPLICATIONS

This application claims the benefit of priority to the filing date ofU.S. Provisional Patent Application Ser. No. 60/263,621, filed Jan. 23,2001.

BACKGROUND OF THE INVENTION

Nucleic acids, proteins and polysaccharides constitute the three majorclasses of biopolymers. While the first two systems are principallylinear assemblies, polysaccharides are structurally more complex. Thisstructural and stereochemical diversity results in a rich content of“information” in relatively small molecules. Nature further “leverages”the structural versatility of polysaccharides by their covalentattachment (i.e., “conjugation”) to other biomolecules such asisoprenoids, fatty acids, neutral lipids, peptides or proteins.Oligosaccharides in the form of glycoconjugates mediate a variety ofevents including inflammation, immunological response, metastasis andfertilization. Cell surface carbohydrates act as biological markers forvarious tumors and as binding sites for other substances includingpathogens.

Proteoglycans are complex protein-carbohydrate assemblies that consistof a core protein and one or more covalently attached glycosaminoglycanchains. For reviews, see: R. V. Iozzo, Annu. Rev. Biochem. 1998, 67,609-652; and M. Bernfield, M. Gotte, P. W. Park, O. Reizes, M. L.Fitzgerald, J. Lincecum, M. Zako, Annu. Rev. Biochem. 1999, 68, 729-777.These linear polysaccharides range in length from ˜20 to 200disaccharide repeat units, each composed of an amino sugar and an uronicacid moiety (FIG. 1).

Heparin-like glycosaminoglycans (HLGAGs) are the most acidic naturallyoccurring biopolymers. These complex polysaccharides, found in theextracellular matrix, play a key role in regulating the biologicalactivity of several proteins in the coagulation cascade along with manyother processes of biomedical importance including growth factorinteractions, virus entry, and angiogenesis. H. E. Conrad, HeparinBinding Proteins; Academic Press 1998. Heparin, isolated from the mastcells of pigs, is currently produced in multi-ton quantities and used ina variety of medical applications. H. Engelberg, Pharmacol. Rev. 1984,36, 91-110. Most prominent is the use of heparin as an anticoagulant inheart disease where it has served as a therapeutic agent since the late1930s. The heterogeneity of heparin results in many severe side effects,making this inexpensive drug dangerous and necessitates closemonitoring. B. H. Chong, Aust. N.Z. J Med. 1992, 22, 145-152.

The heparin-antithrombin III (AT-III) interaction is responsible forheparin's anticoagulant activity and is the only system where the exactsequence of heparin that associates with the protein has beenidentified. Extensive structure-activity studies using syntheticoligosaccharides (M. Petitou, P. Duchaussoy, G. Jaurand, F. Gourvenec,I. Lederman, J.-M. Strassel, T. Barzu, B. Crepon, J.-P. Herault, J.-C.Lormeau, A. Bernat, J.-M. Herbert, J Med. Chem. 1997, 40, 1600-1607; andS. Koshida, Y. Suda, M. Sobel, J. Ormsby, S. Kusumoto, Bioorg. Med.Chem. Lett. 1999, 9, 3127-3132.) as well as NMR (M. Iacomini, B. Casu,M. Guerrini, A. Naggi, A. Pirola, G. Torri, Anal. Biochem. 1999, 274,50-58.) and X-ray crystallography (S. Faram, R. E. Hileman, J. R. Fromm,R. J. Linhardt, D. C. Rees, Science 1996, 271, 1116-1120.) have beenperformed. Based on these studies, a concerted drug development efforthas been undertaken, resulting in the development of a syntheticpentasaccharide heparin analog for use in humans. M. Petitou, P.Duchaussoy, P. A. Driguez, G. Jaurand, J. P. Herault, J. C. Lormeau, C.A. A. van Boeckel, J. M. Herbert, Angew. Chem. Int. Ed. 1998, 37,3009-3014, Angew. Chem. 1998, 110, 3186-3191; and B. Mulloy, M. J.Forster, Glycobiology 2000, 10, 1147-1156. With the exception of theAT-III-heparin interaction, the relationship between structure andfunction of HLGAGs is still poorly understood due to the complexity andheterogeneity of these polymers. Defined HLGAG oligosaccharidesconstitute valuable molecular tools to gain a detailed understanding ofthe sequences of HLGAGs responsible for binding to a particular proteinand modulating its biological activity. For reviews, see: R. V. Iozzo,Annu. Rev. Biochem. 1998, 67, 609-652; and M. Bernfield, M. Gotte, P. W.Park, O. Reizes, M. L. Fitzgerald, J. Lincecum, M. Zako, Annu. Rev.Biochem. 1999, 68, 729-777. Determination of the structure-activityrelationships of HLGAGs will create an opportunity for the discovery ofnovel therapeutic interventions for many disease states.

Over the past two decades, a variety of synthetic methods directed atthe preparation of HLGAG oligosaccharides have been disclosed and heroictotal syntheses (P. Sinay, J.-C. Jacquinet, Carbohydr. Res. 1984, 132,C5-C9; M. Petitou, P. Duchaussoy, I. Lederman, J. Choay, P. Sinay, J.-C.Jacquinet, D. Iorri, Carbohydr. Res. 1986, 147, 221-236.) have resultedin the assembly of AT-III-binding HLGAG oligosaccharides. C. A. A. vanBoeckel, M. Petitou, Angew. Chem. Int. Ed. Engl. 1993, 32, 1671-1690,Angew. Chem. 1993, 105, 1741-1761; P. Westerduin, J. E. M. Basten, M. A.Broekhoven, V. de Kimpe, W. H. A. Kuijpers, C. A. A. van Boeckel, Angew.Chem. Int. Ed. Engl. 1996, 35, 331-333, Angew. Chem. 1996, 108, 339-342.More recently, longer oligosaccharide HLGAG analogs exhibitingimpressive biological activity have been prepared using simplifiedsyntheses. M. Petitou, J.-P. Herault, A. Bernat, P.-A. Driguez, P.Duchaussoy, J.-C. Lormeau, J.-M. Herbert, Nature, 1999, 398, 417-422.Still, the procurement of specific HLGAG sequences required thedevelopment of a new total synthesis stratgey for each oligosaccharidetarget.

Moreover, many additional physiologically-important recognitionphenomena involving carbohydrates have been discovered in recent years.Lectins, proteins which contain carbohydrate recognition domains, havebeen identified. Prominent members of the calcium dependent (C-type)lectin family (Drickamer, K. Curr. Opin. Struct. Biol. 1993, 3, 393) arethe selectins which play a crucial role in leukocyte recruitment ininflammation. Bevilacqua, M. P.; Nelson, R. M. J. Clin. Invest. 1993,91, 379. Members of the C-type lectin superfamily have been described onNK cells and Ly-49, NKR-P1 and NKG2 constitute group V of C-typelectins. While many lectins have been purified and cloned, their ligandshave not been identified due to the heterogeneous nature ofcarbohydrates.

The recognition that interactions between proteins and carbohydrates areinvolved in a wide array of biological recognition events, includingfertilization, molecular targeting, intercellular recognition, andviral, bacterial and fungal pathogenesis, underscores the importance ofcarbohyrates in biological systems. It is now widely appreciated thatthe oligosaccharide portions of glycoproteins and glycolipids mediatecertain recognition events between cells, between cells and ligands,between cells and the extracellular matrix, and between cells andpathogens. See, e.g., U.S. Pat. No. 4,916,219 (describingoligosaccharides with heparin-like anticomplement activity).

These recognition phenomena may be inhibited by oligosaccharides havingthe same sugar sequence and stereochemistry found on the active portionof a glycoprotein or glycolipid involved in the recognition phenomena.The oligosaccharides are believed to compete with the glycoproteins andglycolipids for binding sites on the relevant receptor(s). For example,the disaccharide galactosyl-β-1-4-N-acetylglucosamine is believed to beone component of the glycoproteins which interact with receptors in theplasma membrane of liver cells. Thus, to the extent that they competewith moieties for cellular binding sites, oligosaccharides and othersaccharide compositions have the potential to open new horizons inpharmacology, diagnosis, and therapeutics.

The growing appreciation of the key roles of oligosaccharides andglycoconjugates in fundamental life sustaining processes has stimulateda need for access to usable quantities of these materials.Glycoconjugates are difficult to isolate in homogeneous form from livingcells since they exist as microheterogeneous mixtures. The purificationof these compounds, when possible, is at best tedious and generallyprovides only very small amounts of the compounds. The travailsassociated with isolation of oligo- and poly-saccharides andglycoconjugates from natural sources present a major motivation for thedevelopment and exploitation of chemical synthesis. See, e.g., U.S. Pat.Nos. 4,656,133; 5,308,460; 5,514,784; and 5,854,391 (describing variousmeans of glycosylating saccharides and peptides).

Intense work on the further development of the use ofbiologically-active oligosaccharides is ongoing within a number offields, including: novel diagnostics and blood typing reagents; highlyspecific materials for affinity chromatography; cell specificagglutination reagents; targeting of drugs; monoclonal antibodies, e.g.,against cancer-associated reagents; antibiotic alternatives, based onthe inhibition with specific oligosaccharides of the attachment ofbacteria and viruses to cell surfaces; and stimulation of the growth ofplants and protection of them against pathogens.

The invention of solid phase peptide synthesis by Merrifield 35 yearsago dramatically influenced the strategy for the synthesis ofbiopolymers. The preparation of structurally defined oligopeptides(Atherton, E.; Sheppard, R. C. Solid phase peptide synthesis: Apractical approach; IRL Press at Oxford University Press: Oxford,England, 1989, pp 203) and oligonucleotides (Caruthers, M. H. Science1985, 230, 281) has benefited greatly from the feasibility of conductingtheir assembly on various polymer supports. The advantages of solidmatrix-based synthesis, in terms of allowing for an excess of reagentsto be used and in the facilitation of purification are now wellappreciated. However, the level of complexity associated with thesynthesis of an oligosaccharide on a polymer support dwarfs thatassociated with the other two classes of repeating biooligomers. First,the need to differentiate similar functional groups (hydroxyl and amino)in oligosaccharide construction is much greater than the correspondingneeds in the synthesis of oligopeptides or oligonucleotides.Furthermore, in these latter two cases, there is no stereoselectionassociated with construction of the repeating amide or phosphate bonds.In contrast, each glycosidic bond fashioned in a growing oligosaccharideensemble constitutes a new locus of stereogenicity.

Combinatorial chemistry has been used in the synthesis of large numbersof structurally distinct molecules in a time and resource-efficientmanner. Peptide, oligonucleotide, and small molecule libraries have beenprepared and screened against receptors or enzymes to identifyhigh-affinity ligands or potent inhibitors. These combinatoriallibraries have provided large numbers of compounds to be screenedagainst many targets for biological activity. Every pharmaceuticalcompany now devotes a major effort to the area of combinatorialchemistry in order to develop new lead compounds in a rapid fashion.

The development of protocols for the solid support synthesis ofoligosaccharides and glycopeptides requires solutions to severalproblems. Of course, considerable thought must be addressed to thenature of the support material. The availability of methods forattachment of the carbohydrate from either its “reducing” or“non-reducing” end would be advantageous. Also, selection of a linkerwhich is stable during the synthesis, but can be cleaved easily whenappropriate, is critical. A protecting group strategy that allows forhigh flexibility is desirable. Most important is the matter ofstereospecific and high yielding coupling reactions.

Combinatorial carbohydrate libraries also hold a tremendous potentialwith regard to therapeutic applications. The key role complexoligosaccharides play in biological processes, such as inflammation,immune response, cancer and fertilization makes them highly attractivetherapeutic targets. The ability to create true oligosaccharidelibraries has the potential to trigger a revolution in the area ofbiopharmaceuticals. For example, the generation of combinatorialcarbohydrate libraries will facilitate the rapid identification ofligands to many carbohydrate binding proteins which are involved in avariety of important biological events including inflammation (Giannis,A. Angew. Chem. Int. Ed. Engl. 1994, 33, 178), immune response (Ryan, C.A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1) and metastasis (Feizi, T.Curr. Opin. Struct. Biol. 1993, 3, 701). Analogs of ligands can help todefine important lectin-ligand interactions. Non-natural ligands can bepowerful inhibitors of carbohydrate-protein binding and will facilitatethe study of cascade-like events involving such interactions.Furthermore, inhibitors of carbohydrate-lectin binding are potentialcandidates for a variety of therapeutic applications.

As stated above, due to the difficulties associated with purification ofglycoconjugates and oligosaccharides from natural sources, chemicalsynthesis may be the only way to procure sufficient amounts of thesestructures for detailed biochemical and biophysical studies.Additionally, combinatorial carbohydrate libraries hold great potentialfor the identification of carbohydrate-based ligands to cellularreceptors. Identification of these molecules will open many new avenuesfor the development of diagnostic tools and therapeutic agents.

SUMMARY OF THE INVENTION

Described is a modular, general synthetic strategy for the preparationin solution and on a solid support of heparin, heparin-likeglycosaminoglycans, glycosaminoglycans and non-natural analogs of eachof them. Additionally, the modular strategy provides the basis for thepreparation of combinatorial libraries and parallel libraries of definedglycosaminoglycan oligosaccharides. The defined glycosaminoglycanstructures may be used in high-throughput screening experiments toidentify carbohydrate sequences that regulate a host of recognition andsignal-transduction processes. The determination of specific sequencesinvolved in receptor binding holds great promise for the development ofmolecular tools which will allow modulation of processes underlyingviral entry, angiogenesis, kidney diseases and diseases of the centralnervous system. Notably, the present invention enables the automatedsynthesis of glycosaminoglycans in much the same fashion that peptidesand oligonucleotides are currently assembled.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts various classes of glycosaminoglycans.

FIG. 2 depicts a modular synthesis of heparin according to the presentinvention.

FIG. 3 depicts the syntheses of various monosaccharide building blocksof the present invention.

FIG. 4 depicts the syntheses of various monosaccharide building blocksof the present invention.

FIG. 5 depicts the syntheses of various monosaccharide building blocksof the present invention.

FIG. 6 depicts the syntheses of various monosaccharide building blocksof the present invention.

FIG. 7 depicts the synthesis of certain glucuronic acid disaccharides ofthe present invention using “locked” uronic acid acceptors.

FIG. 8 depicts the synthesis of certain iduronic acid disaccharides ofthe present invention using “locked” uronic acid acceptors.

FIG. 9 depicts the synthesis of certain glucuronic acid disaccharidedonors of the present invention.

FIG. 10 depicts various disaccharide modules of the present inventionthat may be exploited to prepare heparin sequences according to methodsof the present invention.

FIG. 11 depicts the synthesis of various reducing-end disaccharides ofthe present invention.

FIG. 12 depicts the synthesis of various tetrasaccharides according tothe methods of the present invention.

FIG. 13 depicts the synthesis of various trisaccharides according to themethods of the present invention.

FIG. 14 depicts the synthesis of a hexasaccharide according to themethods of the present invention.

FIG. 15 depicts protecting group modification of a tetrasaccharideaccording to the methods of the present invention.

FIG. 16 depicts protecting group modification, sulfation, and the finaldeprotection of a tetrasaccharide according to the methods of thepresent invention.

FIG. 17 depicts a solid-phase synthesis of various glycosaminoglycansaccording to the modular approach of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Overview of the Present Invention

Described is a modular, general synthetic strategy for the preparationin solution and on a solid support of heparin, heparin-likeglycosaminoglycans, glycosaminoglycans and non-natural analogs of eachof them. Additionally, the modular strategy provides the basis for thepreparation of combinatorial libraries and parallel libraries of definedglycosaminoglycan oligosaccharides. The defined glycosaminoglycanstructures may be used in high-throughput screening experiments toidentify carbohydrate sequences that regulate a host of recognition andsignal-transduction processes. The determination of specific sequencesinvolved in receptor binding holds great promise for the development ofmolecular tools which will allow modulation of processes underlyingviral entry, angiogenesis, kidney diseases and diseases of the centralnervous system. Notably, the present invention enables the automatedsynthesis of glycosaminoglycans in much the same fashion that peptidesand oligonucleotides are currently assembled.

Glycosaminoglycans are hetereogeneous polysaccharides that are anchoredin the cellular membrane and constitute a major portion of theextracellular matrix. Heparin, dermatan, hyaluronic acid, and keratanall differ in the type of sugars that are part of the polysaccharideand/or the type of linkage that connects these sugars. See FIG. 1.Although syntheses of short oligosaccharide segments of each type ofglycosaminoglycans have been described previously, most of thesesyntheses were extremely laborious and required a distinct totalsynthesis strategy for each structure targeted. In contrast, theapproach of the present invention is based on the assembly of anyglycosaminoglycan from a limited set of disaccharide building blocks, ina modular fashion that may be carried out on solid support. The methoddescribed here lends itself particularly well to synthesis automationand the preparation of combinatorial libraries and parallel libraries ofdefined oligosaccharides.

Modular Synthesis Strategy of the Present Invention

The structural complexity of HLGAG oligosaccharides necessitates aflexible synthetic approach. Such an approach allows for the preparationof a wide variety of defined structures without requiring the redesignof the synthesis. Therefore, a highly convergent, fully modularsynthetic plan was devised to maximize flexibility and to minimize thenumber of transformations required to fashion an oligosaccharideproduct. A modular, highly convergent synthetic approach for the rapidassembly of defined HLGAG oligosaccharide sequences and libraries ofdefined glycosaminoglycans and non-natural analogs is described. Such anapproach required careful consideration of the many synthetic challengespresented by the great diversity of native structures. The sulfationpatterns mandated the placement of specific protecting groups in allpositions to carry sulfates and different protection on hydroxyls thatremain unaltered. The amine portion of the glucosamine component hasbeen found to be acetylated, sulfated and to exist as the free amine (D.Shukla, J. Liu, P. Blaiklock, N. W. Shworak, X. M. Bai, J. D. Esko, G.H. Cohen, R. J. Eisenberg, R. D. Rosenberg, P. G. Spear, Cell 1999, 99,13-22), thus requiring a protecting group scheme that allows for thedifferentiation of this position.

In addition to the installation of a host of protective groups, thecreation of the glycosidic linkages making up the backbone of HLGAGsposed several challenges. The use of uronic acid derivatives asglycosidating agents had received little attention (C. Tabeur, F.Machetto, J.-M. Mallet, P. Duchaussoy, M. Petitou, P. Sinay, Carbohydr.Res. 1996, 281, 253-276; C. Krog-Jensen, S. Oscarson, Carbohydr. Res.1998, 308, 287-296) and was often circumvented (C. Tabeur, J.-M. Mallet,F. Bono, J.-M. Herbert, M. Petitou, P. Sinay, Bioorg. Med. Chem. Lett.1999, 7, 2003-2012; M. Haller, G.-J. Boons, J. Chem. Soc., Perkin Trans1 2001, 814-822) due to the inherent low reactivity imposed by the C5ester. Stereocontrol during the fashioning of the α-glucosamine linkagewas difficult as anchimeric assistance cannot be exploited thusresulting in the formation of mixtures of glycosides. M. Haller, G.-J.Boons, J. Chem. Soc., Perkin Trans 1 2001, 814-822. Separation of suchanomeric mixtures often necessitated very difficult chromatographicsteps. Finally, the preparation of iduronic acid monosaccharidesrequired lengthy synthetic procedures.

The cornerstone of the modular approach described here is its highconvergency that allows for the preparation of large numbers ofoligosaccharides from a very limited number of fully functionalizedmonosaccharide building blocks. See FIG. 2. Using heparin as an example,four differentially protected glucosamine building blocks can becombined with one glucuronic acid building block and one iduronic acidbuilding block to form eight different disaccharide modules.Modification on the C2 position to allow for introduction of either theC2 hydroxyl or a C2 sulfate and modification of the C2′ amine asN-acetate, N-sulfate or free amine results in a total of 48 differentbuilding blocks that can be combined in a two-step coupling,deprotection cycle to fashion defined oligosaccharides in a highlyconvergent fashion. Further elaboration has been achieved to includetrisaccharide building blocks.

Disclosed is a novel, convergent synthesis of the glucosaminederivatives from glucosamine as common precursor. New syntheticprotocols for the preparation of uronic acid glycosyl donors are alsodislcosed. The generation of the disaccharide modules relies on a newdesign feature for the control of stereochemistry during glycosideformation. By constraining the conformation of the glycosyl acceptorexclusively the desired disaccharides are obtained as a single anomer.

The fully protected HLGAG oligosaccharides are assembled by coupling adisaccharide donor to an acceptor that may be a disaccharide or a longeroligosaccharide. The synthesis of the growing oligosaccharide chainrelies on the formation of β-(1→4) uronic acid linkages and makes use ofa C2 participating group to install this linkage with completestereoselectivity.

Synthesis of Glucosamine Building Blocks of the Present Invention via aCommon Precursor

The first challenge to be tackled in trying to reduce to practice thegeneral approach to HLGAG synthesis was the procurement of large amountsof differentially protected monosaccharide building blocks. In order toaccess large quantities of all monosaccharides, a convergent synthesisfrom inexpensive starting materials was needed that might be performedon large scale with a minimal number of chromatographic purificationsteps. While a host of synthetic methods for the preparation ofglucosamine donors has been explored previously (J. Debenham, R.Rodebaugh, B. Fraser-Reid, Liebigs Ann./Recueil 1997, 791-802.), wefocused on an approach that allowed access to all glucosamine monomersfrom a limited number of advanced intermediates. To identify thebuilding blocks most suitable for installation of the desiredα-glucosamine linkage, different anomeric leaving groups replaced ananomeric silyl ether during the late stages of the synthesis.

A set of glucosamine building blocks was prepared from glucosamine 1.See FIG. 3. Conversion of the 2-amino group into the correspondingazide, necessary for α-selective glycosylations, was followed byacetylation and anomeric silylation to afford crystalline 2 in 72% yieldover four steps. P. B. Alper, S.-C. Hung, C.-H. Wong, Tetrahedron Lett.1996, 37, 6029-6032; A. Vasella, C. Witzig, J.-L. Chiara, M.Martin-Lomas Helv. Chim. Acta, 1991, 74, 2073-2077; and B. La Ferla, L.Lay, M. Guerrini, L. Poletti, L. Panza, G. Russo, Tetrahedron 1999, 55,9867-9880. Deacetylation and installation of the 4,6-benzylidene acetalfurnished common precursor 3. Benzylation of the 3-hydroxyl to fashion 4or acetylation to afford 5 was followed by either removal of the4,6-benzylidene protecting group and installation of 6-acetates, orselective opening of the benzylidene acetal to furnish 6-benzyl groups.These maneuvers provided access to the skeleton of four glucosaminebuilding blocks containing 4-O-silyl ethers as temporary protectinggroups. These TBS groups would later be removed during the preparationof oligosaccharides utilizing the disaccharide modules.

With the desired protecting group patterns in place, we turned ourattention to the installation of different anomeric leaving groups. Lessreactive glycosyl fluoride 10, glycosyl bromide 12, exhibitingintermediate reactivity, and highly reactive glycosyltrichloroacetimidates 11 and 13-15 were prepared for couplings withuronic acid acceptors.

In addition to glucosamine units that act as acceptors duringoligosaccharide formation, glucosamine ‘cap’ monosaccharides wererequired to mark the non-reducing end of the target oligosaccharide. A4-O-benzyl ether was readily introduced by dibenzylation of diol 16followed by transformation into glycosyl trichloroacetimidate 18. SeeFIG. 4. Using this approach, three other cap building blocks withdifferent permutations of acetates and benzyl groups were prepared.

Preparation of Glucuronic Acid and Iduronic Acid Building Blocks of thePresent Invention

After reducing to practice our routes for the preparation of glucosaminebuilding blocks, a reliable route for the synthesis of glucuronic andiduronic acid units was developed. Traditionally, the preparation ofiduronic acid building blocks has been particularly difficult because nodirect precursor may be obtained from natural sources. J. M. J.Tronchet, G. Zosimo-Landolfo, F. Villedon-Denaide, M. Balkadjian, D.Cabrini, F. Barbalat-Rey J. Carbohydr. Chem. 1990, 9, 823-835; and A. B.Smith, R. A. Rivero, K. J. Hale, H. A. Vaccaro J. Am. Chem. Soc. 1991,113, 2092-2112. Efficiency, scalability and the avoidance of excessivechromatography was mandatory for the procurement of large amounts ofthese starting materials. Under this premise, we developed a route todifferentially protected glucuronic acid and iduronic acidmonosaccharides via a common intermediate. See FIG. 5. Commerciallyavailable diacetone glucose 19 was converted to crystalline glucuronicacid furanoside 20 via an eight step procedure that was easily scalableto 100 g starting material and did not require any purification. N. M.Spijker, P. Westerduin, C. A. A. van Boeckel Tetrahedron, 1992, 48,6297-6316; and W. M. Macindoe, H. Ijima, Y. Nakahara, T. OgawaCarbohydr. Res. 1995, 269, 227-257. Access to iduronic acid furanoside22 was readily achieved by inversion of the C5 stereocenter of thetriflate derived from 20. Treatment of 20 and 22 with trifluoroaceticacid resulted in deprotection and formation of the uronic acidpyranosides 21 and 23.

Synthesis of Disaccharide Building Blocks of the Present Invention—TheConcept of “Locked” Acceptors

The installation of α-glucosamine linkages, which are ubiquitous innature (see A. Varki Glycobiology 1993, 3, 97-130.), is a centralfeature of the modular approach to glycosaminoglycans described here.Specifically, we have discovered that 1,2-cyclic acetal protectinggroups may be used to constrain the conformation of glucuronic acidacceptors and to lock the C4 hydroxyl group in an axial position. Thisconformational locking of the glycosyl acceptor results in completelyselective reactions with glycosidating agents, affording disaccharidemodules that previously were only available as anomeric mixtures.

Differentially protected uronic acid monomers (24, 26, 28, 30) wereprepared from triols 21 and 23 by formation of isoproylidene acetals (J.Celas, D. Horton Heterocycles, 1981, 16, 1587-1601; and M. L. Wolfrom,A. B. Diwadkar, J. Gelas, D. Horton Carbohydr. Res.1974, 35, 87-96) orcyclopentylidene acetals (J. M. J. Tronchet, G. Zosimo-Landolfo, F.Villedon-Denaide, M. Balkadjian, D. Cabrini, F. Barbalat-Rey J.Carbohydr. Chem. 1990, 9, 823-835; and A. B. Smith, R. A. Rivero, K. J.Hale, H. A. Vaccaro J. Am. Chem. Soc. 1991, 113, 2092-2112) via reactionwith 2-methoxypropene or methoxycyclopentene under kinetic control. SeeFIG. 6. In addition to the desired compounds, the correspondingfuranosides (25, 27, 29, 31) were obtained and were resubmitted to 1,2acetal formation after cleavage of the acetal protective group.

Union of glycosyl trichloroacetimidate and glycosyl fluoride glucosaminebuilding blocks with glucuronic and iduronic acid glycosyl acceptorsresulted exclusively in the formation of α-linked disaccharides in goodyield. See FIG. 7. Notably, the identities of the cyclic protectinggroup (isopropylidene or cyclopentylidene) and anomeric leaving groupdid not influence the selectivity of the coupling reaction. The complete(α-selectivity of the coupling reactions greatly simplified access todisaccharide modules for heparin assembly and purification of thereaction products. In addition to their use as molecular locks,1,2-acetals are convenient for differential protection ofmonosaccharides and were applied to iduronic acid acceptors. Coupling ofglycosyl donors 18 and 38 with iduronic acid acceptors 28 and 30furnished disaccharides 39-41. After the cyclic acetal protecting groupsserved their purpose they were readily removed to yield disaccharidediols 34, 37, 42, and 43. See FIG. 8.

Subsequent Modification at the Disaccharide Level. After theα-glucosamine linkages had been stereoselectively formed, the resultingdisaccharides had to be converted into competent glycosyl donors and C2participating groups had to be introduced in the uronic acid portion. Inlight of uronic acid C2 hydroxyl or sulfate groups, participatingprotective groups orthogonal to acetates were needed. Levulinoyl groups(Lev) (N. M. Spijker, P. Westerduin, C. A. A. van Boeckel Tetrahedron,1992, 48, 6297-6316; and W. M. Macindoe, H. Ijima, Y. Nakahara, T. OgawaCarbohydr. Res. 1995, 269, 227-257), allyloxycarbonate groups (Alloc)(E. Gentil, M. Potier, P. Boullanger, G. Descotes Carbohydr. Res. 1990,197, 75-91; and T. M. Slaghek, Y. Nakahara, T. Ogawa, J. P. Kamerling,J. F. G. Vliegenthart Carbohydr. Res. 1994, 255, 61-85), andmonochloroacetate groups (MCA) (C.-H. Wong, X.-S. Ye, Z. Zhang J. Am.Chem. Soc. 1998, 120, 7137-7138; and S. Canevari, D. Colombo, F.Compostella, L. Panza, F. Ronchetti, G. Russo, L. Toma Tetrahedron 1999,55, 1469-1478) were installed in a variety of disaccharides to bereplaced by permanent benzyl ether protection when desired. Theselective introduction of 2-hydroxyl protective groups was accomplishedvia two different routes. See FIG. 9. Diacetylation of disaccharidediols 34 and 37 and selective cleavage of the anomeric MCA group wasfollowed by conversion to the corresponding glycosyltrichloroacetimidates 46 and 47. D. Tailler. J.-C. Jacquinet, J.-M. BeauJ. Chem. Soc., Chem. Commun. 1994, 1827-1828; and B. Ernst, G. W. Hart,P. Sinaÿ, Carbohydrates in Chemistry and Biology, Vol. 1, ch. 2,Wiley-VCH, Weinheim, 2000. Iduronic acid containing disaccharide modules59 and 60 were prepared from 42 and 43. For protecting groups that didnot allow for selective anomeric cleavage, anomeric silylation (Z.-H.Jiang, R. R. Schmidt Liebigs Ann. Chem. 1994, 645-651), protection ofthe 2-hydroxyl group, desilylation and preparation of the glycosyltrichloroacetimidates furnished disaccharide modules 53-56.

The second site for modification of the glucosamine residues, asmandated by the naturally occurring glycosaminoglycans structures, isthe C2 amine moiety. Free amines, acetylated amines and sulfated aminesare found in heparin glycosaminoglycans. Therefore, different protectinggroups such as azides, Cbz or Troc groups were introduced todifferentiate this site. The modification of the eight disaccharidebuilding blocks gives rise to a total of 48 differentially protecteddisaccharide modules. See FIG. 10.

Synthesis of Defined Heparin Oligosaccharide Sequences According to theMethods of the Present Invention

Oligosaccharide Assembly Using Disaccharide Modules. Access to theaforementioned set of 48 different disaccharide building blocks providedthe foundation for the preparation of defined heparin structures via arelatively simple two-step coupling-deprotection sequence. In general, adisaccharide glycosyl donor may be coupled with a designated hydroxylgroup of a disaccharide acceptor to form a new glycosidic linkagetherebetween. Different reducing end modules were created by reaction ofany disaccharide with 1-pentenol. See FIG. 11. For example, coupling ofdisaccharide donors 59 and 60 with reducing end modules 62 and 64furnished tetrasaccharides 67-69 in excellent yield. Removal of the4-silyl ether protecting group in the tetrasaccharides rendered themacceptors for further elongation (70-72). See FIG. 12.

Oligosaccharide Assembly Using Trisaccharide Modules. The modularsynthesis of heparin oligosaccharides was further expanded to usetrisaccharide modules, allowing access to all possible structures. Theoverall strategy remained efficient as the linkage between glucosamineand iduronic acid can be established selectively and in high yield. SeeFIG. 2. Trisaccharide modules were readily prepared from the set ofdisaccharides as exemplified by the synthesis of 74 and 76. See FIG. 13.The resulting trisaccharides could be readily converted into acceptors(e.g. 75) or donors (e.g. 76) for the modular assembly ofoligosaccharides. For example, this approach was successfully applied tothe synthesis of hexasaccharide 78 by coupling trisaccharide modules 75and 77 in 62% yield. See FIG. 14.

Sulfation and Final Deprotection. Following the elongation steps, aseries of deprotection and sulfation steps were carried out to createthe desired fully functionalized glycsoaminoglycan by installation ofthe desired sulfation patterns. From the outset of the synthesis,acetates marked positions to be sulfated, whereas benzyl ethersdesignated free hydroxyl groups. C. Tabeur, J.-M. Mallet, F. Bono, J.-M.Herbert, M. Petitou, P. Sina{umlaut over (y )} Bioorg. Med. Chem. 1999,7, 2003-2012. The placement of different C2 protection groups on uronicacid donors was illustrated. Tetrasaccharides 70 and 71 served todemonstrate the final deprotection and sulfation steps. See FIGS. 15 and16. Selective removal of the monochloroacetate group in 70 (V. PozsgayJ. Org. Chem 1998, 63, 5983-5999) and levulinoyl group in 71 (N.Spijker, P. Westerduin, C. A. A. van Boeckel Tetrahedron 1992, 30,6297-6316; and W. M. Macindoe, H. Ijima, Y. Nakahara, T. OgawaCarbohydr. Res. 1995, 269, 227-257.) was achieved in high yield.Permanent protection of the free hydroxyl group was readily accomplishedby benzylation to furnish 72. Alternatively, after saponification of 73,the unprotected hydroxyl groups could be sulfated by reaction withEt₃NSO₃. C. Tabeur, J.-M. Mallet, F. Bono, J.-M. Herbert, M. Petitou, P.Sina{umlaut over (y )} Bioorg. Med. Chem. 1999, 7, 2003-2012. Cleavageof all benzyl ether protective groups and selective N-sulfationfurnished fully functionalized heparin tetrasaccharide 74. C. Tabeur,J.-M. Mallet, F. Bono, J.-M. Herbert, M. Petitou, P. Sina{umlaut over (y)} Bioorg. Med. Chem. 1999, 7, 2003-2012.

Solid-Phase Synthesis of Defined Heparin Oligosaccharide SequencesAccording to the Methods of the Present Invention

The preparation of structurally defined oligopeptides andoligonucleotides has benefited greatly from the feasibility ofconducting their assembly on solid supports. The advantages of solidmatrix based synthesis, in terms of allowing for an excess of reagentsto be used and in their facilitation of purification, are now wellappreciated. However, the level of complexity associated with thesynthesis of oligosaccharides on a polymer support dwarfs thatassociated with the other two classes of repeating biooligomers. Overthe past five years, various strategies and glycosylating agents havebeen employed in the solid-phase synthesis of oligosaccharides. Whilethe number of couplings that may be carried on the solid support islimited, solid-phase synthesis holds particular potential with regard tothe parallel or combinatorial synthesis of diverse sets ofoligosaccharides.

The modular synthesis of heparin and other glycosaminoglycans wasreadily adapted to the solid phase assembly. Following the schemedeveloped by our own laboratory (Plante, O. J.; Palmacci, E. R.;Seeberger, P. H.; Automated Solid-Phase Synthesis of Oligosaccharides;Science 2001, 291, 1523-1527) a polystyrene-bound octenediol linker 82connects the first disaccharide to the solid support to form 83. SeeFIG. 17. Upon removal of the C4 protective group, the sugar may functionagain as a glycosyl acceptor in further couplings until a hexasaccharideof the type 85 is formed. Deprotection and partial sulfation followsolution phase protocols to fashion 86. Cleavage from the solid supportwas accomplished by cross-methathesis, yielding hexasaccharide 87 thatis then fully deprotected and sulfated to furnish hexasaccharide 88.Accordingly, the solid phase method allows for the rapid assembly ofdefined heparin oligosaccharides, as well as the parallel synthesis ofheparin libraries on solid support.

Methods of Administration of Compounds of the Present Invention

Certain of the glycosaminoglycans of the instant invention will beuseful in therapeutic applications, e.g., for treating or preventing avariety of diseases, including cancer, inflammation, and diseases causedor exacerbated by platelet aggregation or angiogenic activity.

Administration of the glycosaminoglycans synthesized via the methods ofthe invention will typically be by routes appropriate forglycosaminoglycan or other carbohydrate compositions, and generallyincludes systemic administration, such as by injection. For example,intravenous injection, such as continuous injection over long timeperiods, can be carried out. Also contemplated are introduction into thevascular system through intraluminal administration or by adventitialadministration using osmotic pumps or implants. Typical implants containbiodegradable materials, such as collagen, polylactate,polylactate/polyglycoside mixtures and the like. These may be formulatedas patches or beads. Typical dosage ranges may be in the range of 0.1-10mg/kg/hr on a constant basis over a period of 5-30 days, preferably7-14, days.

Other acceptable modes of administration include subcutaneous injection,e.g., transmembrane or transdermal or other topical administration forlocalized injury. Localized administration through a continuous releasedevice, such as a supporting matrix, perhaps included in a vasculargraft material, can be useful where the location of the trauma isaccessible.

Formulations suitable for the foregoing modes of administration areknown in the art, and a suitable compendium of formulations is found inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., latest edition.

The glycosaminoglycans may also be labeled using typical methods, suchas radiolabeling, fluorescent labeling, chromophores or enzymes,enabling assays of the amount of such compounds in a biological samplefollowing its administration.

DEFINITIONS

For convenience, before further description of the present invention,certain terms employed in the specification, examples, and appendedclaims are collected here.

The term “nucleophile” is recognized in the art, and as used hereinmeans a chemical moiety having a reactive pair of electrons.

The term “electrophile” is art-recognized and refers to chemicalmoieties which can accept a pair of electrons from a nucleophile asdefined above, or from a Lewis base. Electrophilic moieties useful inthe method of the present invention include halides and sulfonates.

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (σ)constant. This well known constant is described in many references, forinstance, J. March, Advanced Organic Chemistry, McGraw Hill BookCompany, New York, (1977 edition) pp. 251-259. The Hammett constantvalues are generally negative for electron donating groups (σ[P]=−0.66for NH₂) and positive for electron withdrawing groups (σ[P]=0.78 for anitro group), σ[P] indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, ketone, aldehyde, sulfonyl,trifluoromethyl, —CN, chloride, and the like. Exemplaryelectron-donating groups include amino, methoxy, and the like.

The term “catalytic amount” is recognized in the art and means asubstoichiometric amount of a reagent relative to a reactant. As usedherein, a catalytic amount means from 0.0001 to 90 mole percent reagentrelative to a reactant, more preferably from 0.001 to 50 mole percent,still more preferably from 0.01 to 10 mole percent, and even morepreferably from 0.1 to 5 mole percent reagent to reactant.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The terms “arylalkyl” and “aralkyl”, as used herein, refer to an alkylgroup substituted with an aryl group. Likewise, the terms“heteroarylalkyl” and “heteroaralkyl”, as used herein, refer to an alkylgroup substituted with a heteroaryl group.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that comprise a double or triple bond, respectively.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromaticmoieties, —CF₃, —CN, or the like. The term “aryl” also includespolycyclic ring systems having two or more rings in which two or morecarbons are common to two adjoining rings (the rings are “fused”)wherein at least one of the rings is aromatic, e.g., the other rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, perimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorous.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a group permittedby the rules of valence.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonylamino” is art recognized and includes a moiety thatcan be represented by the general formula:

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

The term “sulfonyl”, as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term “sulfoxido” as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₇, m and R₇ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms, and dba represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl, methanesulfonyl, and dibenzylideneacetone,respectively. A more comprehensive list of the abbreviations utilized byorganic chemists of ordinary skill in the art appears in the first issueof each volume of the Journal of Organic Chemistry; this list istypically presented in a table entitled Standard List of Abbreviations.The abbreviations contained in said list, and all abbreviations utilizedby organic chemists of ordinary skill in the art are hereby incorporatedby reference.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The phrase “protecting group” as used herein means temporarymodifications of a potentially reactive functional group which protectit from undesired chemical transformations. Examples of such protectinggroups include esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described hereinabove. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalencies of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

A “polar solvent” means a solvent which has a dielectric constant (ε) of2.9 or greater, such as DMF, THF, ethylene glycol dimethyl ether (DME),DMSO, acetone, acetonitrile, methanol, ethanol, isopropanol, n-propanol,t-butanol or 2-methoxyethyl ether. Preferred solvents are DMF, DME, NMP,and acetonitrile.

A “polar, aprotic solvent” means a polar solvent as defined above whichhas no available hydrogens to exchange with the compounds of thisinvention during reaction, for example DMF, acetonitrile, diglyme, DMSO,or THF.

An “aprotic solvent” means a non-nucleophilic solvent having a boilingpoint range above ambient temperature, preferably from about 25° C. toabout 190° C., more preferably from about 80° C. to about 160° C., mostpreferably from about 80° C. to 150° C., at atmospheric pressure.Examples of such solvents are acetonitrile, toluene, DMF, diglyme, THFor DMSO.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Certain Compounds of the Present Invention

In certain embodiments, the present invention relates to a disaccharideselected from the group consisting of:

wherein

-   -   X represents independently for each occurrence hydroxyl,        acyloxy, silyloxy, halide, alkylthio, arylthio, alkoxy, aryloxy,        or —OC(NH)CCl₃;    -   R represents independently for each occurrence H, alkyl, aryl,        arylalkyl, heteroarylalkyl, silyl, acyl, alkenyloxycarbonyl, or        aralkyloxycarbonyl; and    -   R′ represents independently for each occurrence H, alkyl, aryl,        arylalkyl, or heteroarylalkyl.

In certain embodiments, the present invention relates to a disaccharideas defined above, wherein X represents fluoro, bromo, 4-pentenyloxy or—OC(NH)CCl₃.

In certain embodiments, the present invention relates to a disaccharideas defined above, wherein R′ represents independently for eachoccurrence alkyl.

In certain embodiments, the present invention relates to a disaccharideas defined above, wherein X represents fluoro, bromo, 4-pentenyloxy or—OC(NH)CCl₃; and R′ represents independently for each occurrence alkyl.

In certain embodiments, the present invention relates to a disaccharideselected from the group consisting of:

In certain embodiments, the present invention relates to a trisaccharideselected from the group consisting of:

wherein

-   -   X represents independently for each occurrence hydroxyl,        acyloxy, silyloxy, halide, alkylthio, arylthio, alkoxy, aryloxy,        or —OC(NH)CCl₃;    -   R represents independently for each occurrence H, alkyl, aryl,        arylalkyl, heteroarylalkyl, silyl, acyl, alkenyloxycarbonyl, or        aralkyloxycarbonyl; and    -   R′ represents independently for each occurrence H, alkyl, aryl,        arylalkyl, or heteroarylalkyl.

In certain embodiments, the present invention relates to a trisaccharideas defined above, wherein X represents fluoro, bromo, 4-pentenyloxy or—OC(NH)CCl₃.

In certain embodiments, the present invention relates to a trisaccharideas defined above, wherein R′ represents independently for eachoccurrence alkyl.

In certain embodiments, the present invention relates to a trisaccharideas defined above, wherein X represents fluoro, bromo, 4-pentenyloxy or—OC(NH)CCl₃; and R′ represents independently for each occurrence alkyl.

In certain embodiments, the present invention relates to a trisaccharideselected from the group consisting of:

wherein

-   -   X is silyloxy or —OC(NH)CCl₃; and    -   R is H or silyloxy.        Certain Methods of the Present Invention

In certain embodiments, the present invention relates to a method ofpreparing a glycosaminoglycan, comprising the step of reacting a firstmono-, di- or tri-saccharide, comprising an activated anomeric carbon,with a second mono-, di- or tri-saccharide, comprising a hydroxyl oramino group, to form an oligosaccharide, comprising a glycosidic linkagebetween said anomeric carbon of said first mono-, di- or tri-saccharideand said hydroxyl or amino group of said second mono-, di- ortri-saccharide.

In certain embodiments, the present invention relates to theaforementioned method of preparing a glycosaminoglycan, wherein thefirst mono-, di- or tri-saccharide is not identical to the second mono-,di- or tri-saccharide.

In certain embodiments, the present invention relates to theaforementioned method of preparing a glycosaminoglycan, wherein neitherthe first mono-, di- or tri-saccharide nor the second mono-, di- ortri-saccharide is covalently linked to a solid support.

In certain embodiments, the present invention relates to theaforementioned method of preparing a glycosaminoglycan, wherein thefirst first mono-, di- or tri-saccharide or the second mono-, di- ortri-saccharide is covalently linked to a solid support.

In certain embodiments, the present invention relates to theaforementioned method of preparing a glycosaminoglycan, furthercomprising the step of cleaving said covalent linkage between saidoligosaccharide and said solid support with an alkene metathesiscatalyst and an alkene.

In certain embodiments, the present invention relates to theaforementioned method of preparing a glycosaminoglycan, furthercomprising the step of sulfating a hydroxyl or amino moiety of saidoligosaccharide.

In certain embodiments, the present invention relates to theaforementioned method of preparing a glycosaminoglycan, furthercomprising the step of removing a hydroxyl or amino protecting groupfrom said oligosaccharide by hydrogenolysis.

In certain embodiments, the present invention relates to a method ofpreparing an oligosaccharide comprising an α-glucosamine glycosidiclinkage, comprising the step of reacting a uronic acid glycopyranosylacceptor, comprising a hydroxyl group at C4 and a cyclic acetalcomprising C1 and C2, with a glycosyl donor, comprising an activatedanomeric carbon and an azide functional group at C2, to form anoligosaccharide comprising an α-glycosidic linkage between said hydroxylgroup of said uronic acid glycopyranosyl acceptor and said anomericcarbon of said glycosyl donor.

In certain embodiments, the present invention relates to theaforementioned method of preparing an oligosaccharide comprising anα-glucosamine glycosidic linkage, wherein said uronic acidglycopyranosyl acceptor is an iduronic acid glycopyranosyl acceptor.

In certain embodiments, the present invention relates to theaforementioned method of preparing an oligosaccharide comprising anα-glucosamine glycosidic linkage, wherein said uronic acidglycopyranosyl acceptor is a glucuronic acid glycopyranosyl acceptor.

In certain embodiments, the present invention relates to theaforementioned method of preparing an oligosaccharide comprising anα-glucosamine glycosidic linkage, wherein said glycosyl donor is aglycosyl fluoride or glycosyl trichloroacetimidate.

In certain embodiments, the present invention relates to theaforementioned method of preparing an oligosaccharide comprising anα-glucosamine glycosidic linkage, wherein said glycosyl donor is aglycosyl fluoride or glycosyl trichloroacetimidate.

In certain embodiments, the present invention relates to theaforementioned method of preparing an oligosaccharide comprising anα-glucosamine glycosidic linkage, wherein said cyclic acetal comprisingC1 and C2 of said uronic acid glycopyranosyl acceptor is anisopropylidene acetal or a cyclopentylidene acetal.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; or (8) nasally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred per cent, this amount will range fromabout 1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and a compound of thepresent invention. In certain embodiments, an aforementioned formulationrenders orally bioavailable a compound of the present invention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; (8) absorbents, such as kaolinand bentonite clay; (9) lubricants, such a talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-shelled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or oral cavity; or (4)intravaginally or intravectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” O and B books, Corvallis, Oreg., U.S.A., 1977).

Overview of Strategies and Methods of Combinatorial Chemistry

In the current era of drug development, high throughput screening ofthousands to millions of compounds plays a key role. High throughputscreening generally incorporates automation and robotics to enabletesting these thousands to millions of compounds in one or morebioassays in a relatively short period of time. This high capacityscreening technique requires enormous amounts of “raw materials” havingimmense molecular diversity to fill available capacity. Accordingly,combinatorial chemistry will play a significant role in meeting thisdemand for new molecules for screening. Once “leads” are identifiedusing high throughput screening techniques, combinatorial chemistry willbe advantageously used to optimize these initial leads (whichanalogs/variants will be tested in the same high throughput screeningassay(s) that identified the initial lead).

A combinatorial library for the purposes of the present invention is amixture of chemically-related compounds which may be screened togetherfor a desired property; said libraries may be in solution or covalentlylinked to a solid support. The preparation of many related compounds ina single reaction greatly reduces and simplifies the number of screeningprocesses which need to be carried out. Screening for the appropriatebiological, pharmaceutical, agrochemical or physical property may bedone by conventional methods.

Several challenges have to be met to prepare combinatorial carbohydratelibraries. Synthetic strategies in which either the glycosyl donor orthe glycosyl acceptor is attached to the solid support will be employed.A wide variety of differentially protected monosaccharide buildingblocks have to be prepared. Efficient glycosylation reactions have to beemployed. The resulting libraries can be screened for lectin bindingwhile still on the solid support or after already being cleaved.

The problem of efficiently generating molecular diversity has beentremendously simplified by the advent of combinatorial chemistry. See,e.g., Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555-600. Thisconcept when combined with solid-phase synthesis presents a powerfultechnique for the rapid construction of structurally diverse librariesof compounds which may be screened against therapeutic targets. Avariety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules. See, for example,Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat.Nos. 5,359,115 and 5,362,899: the Ellman U.S. Pat. No. 5,288,514: theStill et al. PCT publication WO 94/08051; Chen et al. (1994) JACS116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092,WO93/09668 and WO91/07087; and the Lerner et al. PCT publicationWO93/20242). Accordingly, a variety of libraries on the order of about16 to 1,000,000 or more diversomers can be synthesized and screened fora particular activity or property.

In an exemplary embodiment, a library of substituted diversomers can besynthesized using the subject reactions adapted to the techniquesdescribed in the Still et al. PCT publication WO 94/08051, e.g., beinglinked to a polymer bead by a hydrolyzable or photolyzable group, e.g.,located at one of the positions of substrate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. In one embodiment, which is particularly suitable for discoveringenzyme inhibitors, the beads can be dispersed on the surface of apermeable membrane, and the diversomers released from the beads by lysisof the bead linker. The diversomer from each bead will diffuse acrossthe membrane to an assay zone, where it will interact with an enzymeassay. Detailed descriptions of a number of combinatorial methodologiesare provided below.

A) Direct Characterization

A growing trend in the field of combinatorial chemistry is to exploitthe sensitivity of techniques such as mass spectrometry (MS), e.g.,which can be used to characterize sub-femtomolar amounts of a compound,and to directly determine the chemical constitution of a compoundselected from a combinatorial library. For instance, where the libraryis provided on an insoluble support matrix, discrete populations ofcompounds can be first released from the support and characterized byMS. In other embodiments, as part of the MS sample preparationtechnique, such MS techniques as MALDI can be used to release a compoundfrom the matrix, particularly where a labile bond is used originally totether the compound to the matrix. For instance, a bead selected from alibrary can be irradiated in a MALDI step in order to release thediversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis

The libraries of the subject method can take the multipin libraryformat. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS81:3998-4002) introduced a method for generating compound libraries by aparallel synthesis on polyacrylic acid-grated polyethylene pins arrayedin the microtitre plate format. The Geysen technique can be used tosynthesize and screen thousands of compounds per week using the multipinmethod, and the tethered compounds may be reused in many assays.Appropriate linker moieties can also been appended to the pins so thatthe compounds may be cleaved from the supports after synthesis forassessment of purity and further evaluation (c.f., Bray et al. (1990)Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166).

C) Divide-Couple-Recombine

In yet another embodiment, a variegated library of compounds can beprovided on a set of beads utilizing the strategy ofdivide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135;and U.S. Pat. Nos 4,631,211; 5,440,016; 5,480,971). Briefly, as the nameimplies, at each synthesis step where degeneracy is introduced into thelibrary, the beads are divided into separate groups equal to the numberof different substituents to be added at a particular position in thelibrary, the different substituents coupled in separate reactions, andthe beads recombined into one pool for the next iteration.

In one embodiment, the divide-couple-recombine strategy can be carriedout using an analogous approach to the so-called “tea bag” method firstdeveloped by Houghten, where compound synthesis occurs on resin sealedinside porous polypropylene bags (Houghten et al. (1986) PNAS82:5131-5135). Substituents are coupled to the compound-bearing resinsby placing the bags in appropriate reaction solutions, while all commonsteps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

D) Combinatorial Libraries by Light-Directed, Spatially AddressableParallel Chemical Synthesis

A scheme of combinatorial synthesis in which the identity of a compoundis given by its locations on a synthesis substrate is termed aspatially-addressable synthesis. In one embodiment, the combinatorialprocess is carried out by controlling the addition of a chemical reagentto specific locations on a solid support (Dower et al. (1991) Annu RepMed Chem 26:271-280; Fodor, S. P. A. (1991) Science 251:767; Pirrung etal. (1992) U.S. Pat. No. 5,143,854; Jacobs et al. (1994) TrendsBiotechnol 12:19-26). The spatial resolution of photolithography affordsminiaturization. This technique can be carried out through the useprotection/deprotection reactions with photolabile protecting groups.

The key points of this technology are illustrated in Gallop et al.(1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared forcoupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers or otherphotolabile linkers. Light is used to selectively activate a specifiedregion of the synthesis support for coupling. Removal of the photolabileprotecting groups by light (deprotection) results in activation ofselected areas. After activation, the first of a set of amino acidanalogs, each bearing a photolabile protecting group on the aminoterminus, is exposed to the entire surface. Coupling only occurs inregions that were addressed by light in the preceding step. The reactionis stopped, the plates washed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized simultaneously; this characteristic leads to thegeneration of many different masking strategies.

E) Encoded Combinatorial Libraries

In yet another embodiment, the subject method utilizes a compoundlibrary provided with an encoded tagging system. A recent improvement inthe identification of active compounds from combinatorial librariesemploys chemical indexing systems using tags that uniquely encode thereaction steps a given bead has undergone and, by inference, thestructure it carries. Conceptually, this approach mimics phage displaylibraries, where activity derives from expressed peptides, but thestructures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. A variety of other forms of encodinghave been reported, including encoding with sequenceable bio-oligomers(e.g., oligonucleotides and peptides), and binary encoding withadditional non-sequenceable tags.

1) Tagging with Sequenceable Bio-Oligomers

The principle of using oligonucleotides to encode combinatorialsynthetic libraries was described in 1992 (Brenner et al. (1992) PNAS89:5381-5383), and an example of such a library appeared the followingyear (Needles et al. (1993) PNAS 90:10700-10704). A combinatoriallibrary of nominally 7⁷ (=823,543) peptides composed of all combinationsof Arg, Gln, Phe, Lys, Val, D-Val and Thr (three-letter amino acidcode), each of which was encoded by a specific dinucleotide (TA, TC, CT,AT, TT, CA and AC, respectively), was prepared by a series ofalternating rounds of peptide and oligonucleotide synthesis on solidsupport. In this work, the amine linking functionality on the bead wasspecifically differentiated toward peptide or oligonucleotide synthesisby simultaneously preincubating the beads with reagents that generateprotected OH groups for oligonucleotide synthesis and protected NH₂groups for peptide synthesis (here, in a ratio of 1:20). When complete,the tags each consisted of 69-mers, 14 units of which carried the code.The bead-bound library was incubated with a fluorescently labeledantibody, and beads containing bound antibody that fluoresced stronglywere harvested by fluorescence-activated cell sorting (FACS). The DNAtags were amplified by PCR and sequenced, and the predicted peptideswere synthesized. Following such techniques, compound libraries can bederived for use in the subject method, where the oligonucleotidesequence of the tag identifies the sequential combinatorial reactionsthat a particular bead underwent, and therefore provides the identity ofthe compound on the bead.

The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesisof non-oligomeric libraries. In certain embodiments, the librariesemploy linkers permitting selective detachment of the test compoundlibrary member for assay.

Peptides have also been employed as tagging molecules for combinatoriallibraries. Two exemplary approaches are described in the art, both ofwhich employ branched linkers to solid phase upon which coding andligand strands are alternately elaborated. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115:2529-2531), orthogonality in synthesisis achieved by employing acid-labile protection for the coding strandand base-labile protection for the compound strand.

In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170),branched linkers are employed so that the coding unit and the testcompound can both be attached to the same functional group on the resin.In one embodiment, a cleavable linker can be placed between the branchpoint and the bead so that cleavage releases a molecule containing bothcode and the compound (Ptek et al. (1991) Tetrahedron Lett32:3891-3894). In another embodiment, the cleavable linker can be placedso that the test compound can be selectively separated from the bead,leaving the code behind. This last construct is particularly valuablebecause it permits screening of the test compound without potentialinterference of the coding groups. Examples in the art of independentcleavage and sequencing of peptide library members and theircorresponding tags has confirmed that the tags can accurately predictthe peptide structure.

2) Non-Sequenceable Tagging: Binary Encoding

An alternative form of encoding the test compound library employs a setof non-sequencable electrophoric tagging molecules that are used as abinary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926). Exemplary tagsare haloaromatic alkyl ethers that are detectable as theirtrimethylsilyl ethers at less than femtomolar levels by electron capturegas chromatography (ECGC). Variations in the length of the alkyl chain,as well as the nature and position of the aromatic halide substituents,permit the synthesis of at least 40 such tags, which in principle canencode 2⁴⁰ (e.g., upwards of 10¹²) different molecules. In the originalreport (Ohlmeyer et al., supra) the tags were bound to about 1% of theavailable amine groups of a peptide library via a photocleavableo-nitrobenzyl linker. This approach is convenient when preparingcombinatorial libraries of peptide-like or other amine-containingmolecules. A more versatile system has, however, been developed thatpermits encoding of essentially any combinatorial library. Here, thecompound would be attached to the solid support via the photocleavablelinker and the tag is attached through a catechol ether linker viacarbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem59:4723-4724). This orthogonal attachment strategy permits the selectivedetachment of library members for assay in solution and subsequentdecoding by ECGC after oxidative detachment of the tag sets.

Although several amide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the electrophoric tags are attacheddirectly to the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031)and provide guidance for generating the subject compound library. Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second,compounds are partially detached and transferred to assay plates; third,a metal binding assay identifies the active wells; fourth, thecorresponding beads are rearrayed singly into new microtiter plates;fifth, single active compounds are identified; and sixth, the structuresare decoded.

Exemplification

The invention may be further understood with reference to the followingexamples, which are presented for illustrative purposes only and whichare non-limiting.

General Experimental Procedures

All chemicals used were reagent grade and used as supplied except whereexpressly noted. Anhydrous methanol (MeOH) and dimethylformamide (DMF)were purchased from Aldrich in SureSeal bottles. Dichloromethane(CH₂Cl₂), diethyl ether, toluene and tetrahydrofuran (THF) werepurchased from J.T. Baker (Cycletainer™) and passed through a neutralalumina column prior to use. Pyridine, 2,4,6 collidine and acetonitrile(CH₃CN) were refluxed over calcium hydride and distilled prior to use.Analytical thin-layer chromatography was performed on E. Merck silicagel 60 F₂₅₄ plates (0.25 mm). Compounds were visualized by dipping theplates in a cerium sulfate-ammonium molybdate solution followed byheating. Liquid column chromatography was performed using forced flow ofthe indicated solvent on Silicycle 230-400 mesh (60 Å pore diameter)silica gel. ¹H NMR spectra were obtained on a Varian VXR-500 (500 MHz)or a Varian-300 (300 MHz) spectrometer and are reported in parts permillion (δ) relative to CHCl₃ (7.27 ppm). Coupling constants (J) arereported in Hertz. ¹³C NMR spectra were obtained on a Varian VXR-500(125 MHz) or a Varian-300 (75 MHz) spectrometer and are reported in δrelative to CDCl₃ (77.23 ppm) as an internal reference.

Specific Experimental Procedures

1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose

Preparation of TfN₃

Dichloromethane was added to a solution of NaN₃ (59.5 g, 0.92 mol) inwater (150 mL) at 0° C. CH₂Cl₂ (250 mL). The mixture was stirredvigorously and treated with trifluoromethanesulfonic anhydride (31.0 mL,0.19 mol) over a period of 3 h at 0° C. After the complete addition oftrifluoromethanesulfonic anhydride, the reaction mixture was stirred at0° C. for 2.5 h. The aqueous phase was extracted with CH₂Cl₂ (2×100 mL)and the combined organic layers washed with saturated Na₂CO₃ and savedfor use in the next step. [Caution: TfN₃ is explosive when not insolution.]

To a solution of glucosamine hydrochloride 1 (20.0 g, 0.092 mol) inwater (300 mL) was added CuSO₄ (140 mg, 0.88 mmol) and K₂CO₃ (19.2 g,0.14 mol). Methanol (600 mL) was added to the reaction mixture followedby the addition of the TfN₃ solution. Methanol was added until thesolution was homogeneous (˜300 mL). The clear blue solution was allowedto stir for 24 h at room temperature. Glycine (70 g) was added and thereaction mixture was again allowed to stir for 24 h. The glycine wasfiltered off and the solvent was removed in vacuo to afford a brown oil.The oil was taken up in pyridine (95 mL), cooled to 0° C. and DMAP (˜30mg) and acetic anhydride (86 mL, 0.91 mol) were added. The solution wasstirred for 12 h at room temperature. The reaction was quenched withsaturated NaHCO₃ and the aqueous phase was extracted with CH₂Cl₂ (3×1000mL). The organic phase was dried over MgSO₄, filtered and the solventwas removed in vacuo to yield a brown oil. Warm ethanol was added untilthe solution was homogeneous. The resulting solution was cooled to −20°C. and a white precipitate formed. Cold water was then added, the whiteprecipitate was filtered and washed with water and cold ethanol toafford β-product (23.8 g, 0.063 mol, 68%) as a colorless solid. ¹H-NMR(300 MHz, CDCl₃) δ 5.55 (d, J=8.6 Hz, 1H), 5.15-5.00 (m, 2H), 4.31 (dd,J=4.6, 12.5 Hz, 1H), 4.08 (dd, J=2.1, 12.5 Hz, 1H), 3.75 (ddd, J=2.1,4.4, 6.3 Hz, 1H), 3.65-3.72 (m, 1H), 2.20 (s, 3H), 2.10 (s, 3H), 2.08(s, 3H), 2.04 (s, 3H); IR (thin film) 2959, 2112, 1747 cm⁻¹. Flashchromatography of the mother liquor (Hexanes:EtOAc 7:3) afforded acolorless oil (mixture of α/β) (5.6 g, 0.015 mmol, 17%). The spectraldata was in agreement with the reported data. (P. B. Alper, S.-C. Hung,C.-H. Wong, Tetrahedron Lett. 1996, 37, 6029-6032; A. Vasella, C.Witzig, J.-L. Chiara, M. Martin-Lomas, Helv. Chim. Acta 1991, 74,2073-2077).

tert-Butyldimethylsilyl3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside 2

1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (30.2 g, 80 mmol)was coevaporated twice with toluene and dissolved in THF and methanol(7:3, 300 mL). The solution was cooled to 0° C. and gaseous anhydrousammonia was bubbled through at a modest rate. After 15 min, nitrogen wasbubbled through the solution to remove excess ammonia and the solventwas removed in vacuo to afford a brown oil. The residue was coevaporatedtwice with toluene and dissolved in CH₂Cl₂ (150 mL). Imidazole (10.9 g,160 mmol) and tert-butyldimethylsilyl chloride (13.3 g, 88 mmol) wereadded. After 2 h, the mixture was diluted with EtOAc, washed with water,1 N HCl (2×) and water. The organic layer was dried over MgSO₄,filtered, and the solvent was removed in vacuo. Crystallization fromethanol afforded 2 (29.8 g, 67 mmol, 84%) as colorless crystals. ¹H-NMR(300 MHz, CDCl₃) δ 5.00-4.90 (m, 2H), 4.63 (d, J=7.6 Hz, 1H) 4.20 (dd,J=5.9, 12.1 Hz, 1H), 4.09 (dd, J=2.6, 12.1 Hz, 1H), 3.70-3.64 (m, 1H),3.48-3.40 (m, 1H), 2.10 (s, 3H), 2.08 (s, 3H), 2.00 (s, 3H), 0.95 (s,9H), 0.15 (s, 6H). The spectral data was in agreement with the reporteddata. (W. Kinzy, R. R. Schmidt, Liebigs Ann. Chem. 1985, 1537-1545; forthe procedure see: B. La Ferla, L. Lay, M. Guerrini, L. Poletti, L.Panza, G. Russo, Tetrahedron 1999, 55, 9867-9880).

tert-Butyldimethylsilyl2-azido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside 3

tert-Butyldimethylsilyl3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside 2 (36.9 g; 82.8mmol) was dissolved in methanol (300 mL) and NaOMe (25% in MeOH, 5.4 mL)was added. After 15 min, DOWEX-50 acidic resin was added and the mixturewas stirred until the pH reached 6. The DOWEX resin was filtered off andthe solvent was removed in vacuo to afford a yellow oil. The residue wascoevaporated twice with acetonitrile and dissolved in acetonitrile (400mL). Benzaldehyde dimethyl acetal (24.8 mL, 165 mmol) andp-toluenesulfonic acid monohydrate (400 mg, 2.1 mmol) were added. Afterstirring overnight at room temperature, triethylamine (5 mL) was addedand the solvents evaporated. Flash chromatography on silica gel(Hexanes:EtOAc 95:5→9:1) afforded 3 (29.0 g, 71.2 mmol, 86%) as acolorless oil. ¹H-NMR (300 MHz, CDCl₃) δ 7.55-7.45 (m, 2H), 7.45-7.38(m, 3H), 5.52 (s, 1H), 4.65 (d, J=7.65 Hz, 1H), 4.29 (dd, J=4.9, 10.4Hz, 1H), 3.77 (t, J=10.1 Hz, 1H), 3.60-3.30 (m, 4H), 2.91 (s, 1H), 1.00(s, 9H), 0.19 (s, 6H). The spectral data was in agreement with thereported data. (C. Murakata, T. Ogawa, Carbohydrate Res. 1992, 234,75-91)

tert-Butyldimethylsilyl2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside 4

tert-Butyldimethylsilyl2-azido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside 3 (28.1 g, 68.97mmol) was dissolved in CH₂Cl₂ (250 mL). Powdered, freshly activated 4 Åmolecular sieves (45 g) and benzyl bromide (20.5 mL, 172 mmol) wereadded and the mixture was stirred for 30 min. Silver(I) oxide (47 g, 203mmol) was added and the reaction vessel was covered in aluminum foil toexclude light. After 8 h, the reaction mixture was filtered throughCelite and the filtrate was concentrated in vacuo. Flash chromatographyon silica (Hexanes:EtOAc 50:1) afforded 4 (32.6 g, 65.5 mmol, 95%) as acolorless solid. ¹H NMR (300 MHz, CDCl₃) 7.60-7.28 (m, 10H), 5.51 (s,1H), 4.98 (d, J=11.5 Hz, 1H), 4.84 (d, J=11.5 Hz, 1H), 4.63 (d, J=7.5Hz, 1H), 4.33 (dd, J=5.0, 9.4 Hz, 1H), 3.89-3.73 (m, 2H), 3.60-3.35 (m,3H), 0.98 (s, 9H), 0.17 (s, 6H). The spectral data was in agreement withthe reported data. (C. Murakata, T. Ogawa, Carbohydrate Res. 1992, 234,75-91).

tert-Butyldimethylsilyl3-O-acetyl-2-azido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside 5

tert-Butyldimethylsilyl2-azido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside 3 (9.5 g, 23.3mmol) was dissolved in CH₂Cl₂ (150 mL) and pyridine (21 mL), DMAP (280mg, 2.3 mmol) and acetic anhydride (10 mL, 106 mol) were added. Thereaction mixture was stirred overnight, water was added and stirred for1 h. The organic layer was extracted with water, 1 N HCl, water andsaturated NaHCO₃. The organic phase was dried over MgSO₄, filtered andthe solvent was removed in vacuo. Flash chromatography on silica gel(Hexanes:EtOAc 50:1→5:1) afforded 5 (9.96 g, 22.1 mmol, 95%) as acolorless crystalline solid. [α]²⁴ _(D): −72.1 (c 0.99, CH₂Cl₂); IR(thin film) 2111, 1751, 1370, 1222, 1099, 841 cm⁻¹; ¹H-NMR (300 MHz,CDCl₃) δ 7.45-7.33 (m, 5H), 5.49 (s, 1H), 5.13 (dd, J=9.7, 9.9 Hz, 1H),4.72 (d, J=7.6 Hz, 1H), 4.31 (dd, J=4.9, 10.5 Hz, 1H), 3.80 (dd, J=10.2,10.3 Hz, 1H), 3.65 (dd, J=9.4, 9.5 Hz, 1H), 3.49 (ddd, J=4.9, 9.6, 9.6Hz, 1H), 3.42 (dd, J=7.5, 10.1 Hz, 1H), 2.14 (s, 3H), 0.95 (s, 9H), 0.19(s, 3H), 0.18 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 169.9, 136.9, 129.3,128.4, 126.3, 101.7, 97.8, 78.9, 71.1, 68.7, 67.4, 66.8, 25.8, 21.2,18.2, −4.1, −4.9; FAB MS (C₂₁H₃₁N₃O₆Si) m/z (M⁺) calcd 449.1982, obsd449.1876.

Synthesis of 6-O-Acetyl Glucosamine Series tert-Butyldimethylsilyl2-azido-3-O-benzyl-2-deoxy-β-D-glucopyranoside

tert-Butyldimethylsilyl2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-β-D-gluco-pyranoside 4(32.6 g, 65.5 mmol) was dissolved in CH₂Cl₂ (1.5 L) and trifluoroaceticacid (60% aq., 54 mL) was added. The resulting mixture was stirredvigorously at room temperature for 8.5 h and saturated NaHCO₃ was addedcarefully. After phase separation, the aqueous layer was extracted withCH₂Cl₂. The combined organic layers were dried over Na₂SO₄, filtered andthe solvents were removed in vacuo. Flash chromatography on silica(Hexanes:EtOAc 4:1→1:1) afforded 7 (25 g, 61 mmol, 92%) as a colorlessoil. [α]²⁴ _(D): −30.4 (c 1.00, CH₂Cl₂); IR (thin film) 3415, 2110,1361, 1079 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.44-7.30 (m, 5H), 4.97 (d,J=11.4 Hz, 1H), 4.72 (d, J=11.4 Hz, 1H), 4.58 (d, J=7.5 Hz, 1H), 3.84(dd, J=3.7, 11.8 Hz, 1H), 3.75 (dd, J=4.8, 11.8 Hz, 1H), 3.59 (dd,J=8.7, 9.5 Hz, 1H), 3.36-3.26 (m, 2H), 3.22 (dd, J=8.6, 9.9 Hz, 1H),0.96 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ138.2, 128.9, 128.34, 128.26, 97.5, 82.5, 75.3, 75.2, 70.7, 68.5, 62.8,25.8, 18.1, −4.0, −4.9; FAB MS (C₁₉H₃₁N₃O₅Si) m/z (M⁺) calcd 409.2033,obsd 409.2029. The spectral data was in agreement with the reported data(A. G. M. Barrett, D. Pilipauskas, J. Org. Chem. 1991, 56, 2787-2800).

tert-Butyldimethylsilyl 3-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside

tert-Butyldimethylsilyl3-O-acetyl-2-azido-4,6-O-benzylidene-2-deoxy-β-D-gluco-pyranoside 5 (9.5g, 21.1 mmol) was dissolved in CH₂Cl₂ (500 mL) and trifluoroacetic acid(60% aq., 17 mL) was added. The resulting mixture was stirred vigorouslyat room temperature overnight and saturated NaHCO₃ was added carefully.After phase separation, the aqueous layer was extracted with CH₂Cl₂. Thecombined organic layers were dried over Na₂SO₄, filtered and thesolvents were removed in vacuo. Flash chromatography on silica gel(Hexanes:EtOAc 4:1→1:1) afforded product (7.25 g, 20 mmol, 95%) as acolorless solid. [α]²⁴ _(D): −26.4 (c 1.00, CH₂Cl₂); IR (thin film)3387, 2112, 1748, 1254, cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 4.77 (dd, J=9.2,9.5 Hz, 1H), 4.64 (d, J=7.6 Hz, 1H), 3.90 (dd, J=3.7, 11.9 Hz, 1H), 3.81(dd, J=4.9, 11.9 Hz, 1H), 3.66 (dd, J=9.5, 9.5 Hz, 1H), 3.41-3.36 (m,1H), 3.34 (dd, J=7.6, 10.4 Hz, 1H), 3.08-2.92 (br s, 1H), 2.15-1.92 (brs, 1H), 2.19 (s, 3H), 0.94 (s, 9H), 0.174 (s, 3H), 0.168 (s, 3H);¹³C-NMR (125 MHz, CDCl₃) δ 172.2, 97.2, 76.0, 75.8, 70.1, 66.2, 62.6,25.7, 21.2, 18.1, −4.1, −5.0; FAB MS (C₁₄H₂₇N₃O₆Si) m/z (M⁺) calcd361.1669, obsd 361.1677.

tert-Butyldimethylsilyl6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-β-D-glucopyranoside 6

Acetyl chloride (4.5 mL, 63.3 mmol) was added dropwise to a solution oftert-butyldimethylsilyl 2-azido-3-O-benzyl-2-deoxy-β-D-glucopyranoside(25 g, 61 mmol) in 2,4,6-collidine (110 mL) under nitrogen at −40° C.After stirring at −40° C. overnight, water was added. The mixture waspoured into EtOAc and extracted with 1 N HCl, brine and saturatedNaHCO₃. The organic phase was dried over Na₂SO₄, filtered and thesolvents were removed in vacuo to afford 6 (26.5 g, 58.7 mmol, 96%) as acolorless solid. [α]²⁴ _(D): −27.1 (c 1.00, CH₂Cl₂); IR (thin film)3482, 2110, 1743, 1255 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.43-7.30 (m,5H), 4.96 (d, J=11.4 Hz, 1H), 4.73 (d, J=11.4 Hz, 1H), 4.55 (d, J=7.6Hz, 1H), 4.34-4.27 (m, 2H), 3.50-3.38 (m, 2H), 3.33 (dd, J=7.6, 9.9 Hz,1H), 3.21 (dd, J=8.3, 9.9 Hz, 1H), 2.59-2.46 (br s, 1H), 2.09 (s, 3H),0.96 (s, 9H), 0.18 (s, 6H); ¹³C-NMR (75 MHz, CDCl₃) δ 171.5, 138.1,128.8, 128.31, 128.28, 97.4, 82.2, 75.3, 73.8, 70.3, 68.3, 63.5, 25.8,21.1, 18.3, −4.1, −5.0; FAB MS (C₂₁H₃₃N₃O₆Si) m/z (M⁺) calcd 451.2138,obsd 451.2135.

tert-Butyldimethylsilyl3,6-di-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside 7

Acetyl chloride (1.5 mL, 21.1 mmol) was added slowly to a solution oftert-butyldimethylsilyl 3-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside(7.25 g, 20 mmol) in 2,4,6-collidine (47 mL) under nitrogen at −40° C.After stirring at −40° C. overnight, water was added. The mixture waspoured into EtOAc and extracted with 1 N HCl, brine and saturatedNaHCO₃. The organic phase was dried over Na₂SO₄, filtered and thesolvents were removed in vacuo. Flash chromatography on silica gel(Hexanes:EtOAc 5:1→4:1) afforded 7 (7.59 g, 18.8 mmol, 94%) as acolorless solid. [a]²⁴ _(D): −31.6 (c 1.00, CH₂Cl₂); IR (thin film onNaCl) 3459, 2112, 1747, 1233, 1042, 841 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ4.81-4.74 (m, 1H), 4.62 (d, J=7.6 Hz, 1H), 4.37-4.31 (m, 2H), 3.75-3.46(m, 2H), 3.53 (dd, J=7.7, 10.3 Hz, 1H), 3.13-3.09 (m, 1H), 2.18 (s, 3H),2.10 (s, 3H), 0.94 (s, 9H), 0.17 (s, 6H); ¹³C-NMR (75 MHz, CDCl₃) δ171.8, 171.5, 97.2, 75.5, 74.3, 69.8, 66.0, 63.4, 25.8, 21.2, 21.1,18.2, −4.2, −5.0; FAB MS (C₁₆H₂₉N₃O₇Si) m/z (M⁺) calcd 403.1775, obsd403.1779.

tert-Butyldimethylsilyl6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside

tert-Butyldimethylsilyl trifluormethanesulfonate (3.4 mL, 14.8 mmol) wasadded to a solution of tert-butyldimethylsilyl6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-β-D-glucopyranoside 6 (5.15 g,11.4 mmol) and 2,6-lutidine (3.3 mL, 28.3 mmol) in CH₂Cl₂ (50 mL) at−20° C. The reaction was allowed to warn to room temperature and stirfor 1 h. The mixture was poured into EtOAc and the aqueous layer wasextracted with 1 N HCl, brine and saturated NaHCO₃. The organic layerwas dried over Na₂SO₄, filtered and the solvents were removed in vacuo.Flash chromatography on silica gel (Hexanes:EtOAc 199:1→98:2) affordedproduct (5.8 g, 10.3 mmol, 90%) as a colorless oil. [α]²⁴ _(D): +36.1 (c1.00, CH₂Cl₂); IR (thin film) 2110, 1748, 1112 cm⁻¹; ¹H-NMR (300 MHz,CDCl₃) δ 7.40-7.28 (m, 5H), 4.93 (d, J=11.1 Hz, 1H), 4.71 (d, J=11.1 Hz,1H), 4.56 (d, J=7.6 Hz, 1H), 4.42 (dd, J=2.2, 11.6 Hz, 1H), 4.07 (dd,J=6.6, 11.6 Hz, 1H), 3.59 (dd, J=8.3, 9.5 Hz, 1H), 3.45 (ddd, J=2.2,6.6, 9.5 Hz, 1H), 3.34 (dd, J=7.6, 9.9 Hz, 1H), 3.20 (dd, J=8.3, 9.9 Hz,1H), 2.08 (s, 3H), 0.94 (s, 9H), 0.89 (s, 9H), 0.164 (s, 3H), 0.160 (s,3H), 0.05 (s, 3H), 0.03 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 170.8, 138.4,128.5, 127.7, 127.6, 97.4, 83.0, 75.2, 74.6, 71.3, 69.2, 63.6, 26.1,25.8, 21.1, 18.3, −3.5, −4.1, −4.6, −5.0; FAB MS (C₂₇H₄₇N₃O₆Si₂) m/z(M⁺) calcd 565.3003, obsd 565.3011.

tert-Butyldimethylsilyl3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside

tert-Butyldimethylsilyl trifluoromethanesulfonate (4.1 mL, 17.9 mmol)was added to a solution of tert-butyldimethylsilyl3,6-di-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside 7 (4.70 g, 11.65mmol) and 2,6-lutidine (3.5 mL, 30 mmol) in CH₂Cl₂ (25 mL) at −20° C.The reaction was allowed to warm to room temperature and stirred for 1h. The mixture was poured into EtOAc and extracted with 1 N HCl, brineand saturated NaHCO₃. The organic layer was dried over Na₂SO₄, filteredand solvents removed in vacuo. Flash chromatography on silica gel(Hexanes:EtOAc 199:1→98:2) afforded product (5.61 g, 10.8 mmol, 93%) asa colorless oil. [α]²⁴ _(D): −3.1 (c 1.00, CH₂Cl₂); IR (thin film) 2111,1750, 1363, 1221 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 4.87 (dd, J=8.8, 10.4Hz, 1H), 4.63 (d, J=7.6 Hz, 1H), 4.39 (dd, J=2.2, 11.7 Hz, 1H), 4.08(dd, J=6.2, 11.7 Hz, 1H), 3.67 (dd, J=9.1, 9.2 Hz, 1H), 3.54-3.46 (m,1H), 3.27 (dd, J=7.6, 10.4 Hz, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 0.92 (s,9H), 0.83 (s, 9H), 0.15 (s, 3H), 0.14 (s, 3H), 0.05 (s, 3H), 0.04 (s,3H); ¹³C-NMR (75 MHz, CDCl₃) δ 170.7, 169.9, 97.1, 74.6, 74.5, 69.6,66.8, 63.1, 25.80, 25.75, 21.6, 21.1, 18.2, 18.1, −3.9, −4.3, −4.6,−5.0; FAB MS (C ₂₂H₄₃N₃O₇Si₂) m/z (M⁺) calcd 517.2639, obsd 517.2635.

Synthesis of 6-O-Benzyl Glucosamine Series tert-Butyldimethylsilyl2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside 8

tert-Butyldimethylsilyl2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside 4 (3.6g, 7.24 mmol) and triethylsilane (6.5 mL, 43.4 mmol) were dissolved inanhydrous CH₂Cl₂ (70 mL) under nitrogen at 0° C. and trifluoroaceticacid (3.3 mL, 43.4 mmol) was added dropwise over 5 min. The reactionmixture was slowly warmed to room temperature, stirred for 5 h andquenched with saturated NaHCO₃. After addition of CH₂Cl₂ and phaseseparation, the aqueous phase was extracted with CH₂Cl₂. The combinedorganic phases were dried over MgSO₄, filtered and the solvents wereremoved in vacuo. Flash chromatography on silica gel (Hexanes:EtOAc8:1→6:1) afforded 8 (3.1 g, 6.2 mmol, 85%) as a colorless oil. [α]²⁴_(D): −33.9 (c 1.00, CH₂Cl₂); IR (thin film) 3472, 2111, 1257, 1113,1069 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.45-7.28 (m, 10H), 4.93 (d, J=11.4Hz, 1H), 4.77 (d, J=11.4 Hz, 1H), 4.61 (d, J=12.1 Hz, 1H), 4.56 (d,J=12.1 Hz, 1H), 4.55 (d, J=7.5 Hz, 1H), 3.73 (d, J=4.8 Hz, 2H), 3.65(dd, J=8.5, 9.7 Hz, 1H), 3.46-3.39 (m, 1H), 3.33 (dd, J=7.5, 10.0 Hz,1H), 3.23 (dd, J=8.5, 10.0 Hz, 1H), 0.95 (s, 9H), 0.18 (s, 6H); ¹³C-NMR(75 MHz, CDCl₃) δ 138.3, 137.9, 128.8, 128.6, 128.23, 128.16, 127.9,127.8, 97.4, 82.5, 75.2, 74.1, 73.9, 72.2, 70.6, 68.3, 25.9, 18.3, −4.0,−5.0; FAB MS (C₂₆H₃₇N₃O₅Si) m/z (M⁺) calcd 499.2502, obsd 499.2513. Thespectral data was in agreement with the reported data (C. Murakata, T.Ogawa, Carbohydrate Res. 1992, 234, 75-91).

tert-Butyldimethylsilyl3-O-acetyl-2-azido-6-O-benzyl-2-deoxy-β-D-glucopyranoside 9

tert-Butyldimethylsilyl3-O-acetyl-2-azido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside 5 (42.0mg, 0.093 mmol) and triethylsilane (75 μL, 0.47 mmol) were dissolved inCH₂Cl₂ (930 μL) under argon at 0° C. and trifluoroacetic acid (36 μL,0.47 mmol) was added dropwise over 6 min. The reaction mixture wasslowly warmed to room temperature, stirred for 3 h and quenched withsaturated NaHCO₃. After addition of CH₂Cl₂ and phase separation, theaqueous phase was extracted three times with CH₂Cl₂. The combinedorganic phases were dried over MgSO₄, filtered and the solvents wereremoved in vacuo. Flash chromatography on silica gel (Hexanes:EtOAc8:1→6:1) furnished 9 (38.1 mg, 91%) as a colorless oil. [α]²⁴ _(D):−21.6 (c 1.00, CH₂Cl₂); IR (thin film) 3434, 2111, 1749, 1252 cm⁻¹;¹H-NMR (300 MHz, CDCl₃) δ 7.40-2.28 (m, 5H), 4.80 (dd, J=9.1 Hz, 10.3Hz, 1H), 4.61 (d, J=7.7 Hz, 1H), 4.61-4.57 (m, 2H), 3.75 (dd, J=1.6, 4.9Hz, 1H), 3.69 (ddd, J=3.5, 9.3, 9.3 Hz, 1H), 3.53-3.45 (m, 1H), 3.36(dd, J=7.7, 10.3 Hz, 1H), 3.00 (d, J=3.7 Hz, 1H), 2.18 (s, 3H), 0.95 (s,9H), 0.178 (s, 3H), 0.175 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 171.5,137.7, 128.6, 128.0, 127.8, 97.3, 75.4, 74.4, 73.9, 71.1, 70.3, 66.1,25.8, 21.3, 18.2, −4.1, −5.0; FAB MS (C₂₁H₃₃N₃O₆Si) m/z (M⁺) calcd451.2138, obsd 451.213 1.

tert-Butyldimethylsilyl2-azido-3,6-di-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside

To a solution of tert-butyldimethylsilyl2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside 8 (353.7 mg, 0.708mmol) and 2,6-lutidine (206 μL, 1.77 mmol) in CH₂Cl₂ (800 μL) was addedtert-butyldimethylsilyl trifluoromethanesulfonate (244 μL, 1.06 mmol)under argon at room temperature. The reaction mixture was stirred for 1h and quenched with saturated NaHCO₃. After addition of CH₂Cl₂ and phaseseparation the aqueous phase was extracted four times with CH₂Cl₂. Thecombined organic phases were dried over MgSO₄, filtered and solventsremoved in vacuo. Flash chromatography on silica gel (Hexanes:EtOAc25:1) afforded product (420 mg, 97%) as a colorless solid. [α]²⁴ _(D):+31.1 (c 1.00, CH₂Cl₂); IR (thin film) 2109, 1472, 1360, 1107, 1066cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.40-7.28 (m, 1H), 4.93 (d, J=11.1 Hz,1H), 4.72 (d, J=1.1 Hz, 1H), 4.64 (d, J=12.2 Hz, 1H), 4.58 (d, J=7.7 Hz,1H), 4.53 (d, J=12.2 Hz, 1H), 3.78-3.64 (m, 2H), 3.59 (dd, J=5.5, 10.8Hz, 1H), 3.44-3.39 (m, 1H), 3.34 (dd, J=8.5, 9.8 Hz, 1H), 3.19 (dd,J=8.5, 9.8 Hz, 1H), 0.96 (s, 9H), 0.87 (s, 9H), 0.20 (s, 3H), 0.19 (s,3H), 0.03 (s, 3H), 0.02 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 138.7, 138.5,128.50, 128.46, 127.63, 127.60, 97.5, 83.4, 76.6, 75.0, 73.5, 71.0,69.3, 69.2, 26.1, 25.8, 18.23, 18.19, −3.6, −4.0, −4.6, −5.0; FAB MS(C₃₂H₅₁N₃O₅Si₂) m/z (M⁺) calcd 613.3367, obsd 613.3359.

tert-Butyldimethylsilyl3-O-acetyl-2-azido-6-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside

tert-Butyldimethylsilyl trifluoromethanesulfonate (9.5 μL, 0.041 mmol)was added to a solution of tert-butyldimethylsilyl3-O-acetyl-2-azido-6-O-benzyl-2-deoxy-β-D-glucopyranoside 9 (12.4 mg,0.027 mmol) and 2,6-lutidine (8.0 μL, 0.069 mmol) in CH₂Cl₂ (200 μL)under argon at room temperature. The reaction mixture was stirred for 1h and quenched with saturated NaHCO₃. After addition of CH₂Cl₂ and phaseseparation, the aqueous phase was extracted three times with CH₂Cl₂. Thecombined organic phases were dried over MgSO₄, filtered and the solventswere removed in vacuo. Flash chromatography on silica gel (Hexanes:EtOAc30:1) afforded product (15.3 mg, 98%) as a colorless solid. [α]²⁴ _(D):−11.3 (c 0.71, CH₂Cl₂); IR (thin film) 2109, 1752, 1473, 1221, 1107, 899cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.38-7.29 (m, 5H), 4.87 (dd, J=9.0, 10.5Hz, 1H), 4.65 (d, J=12.4 Hz, 1H), 4.64 (d, J=7.8 Hz, 1H), 4.53 (d,J=12.4 Hz, 1H), 3.79 (dd, J=9.3, 9.1 Hz, 1H), 3.70-3.60 (m, 2H),3.45-3.38 (m, 1H), 2.15 (s, 3H), 0.95 (s, 9H), 0.82 (s, 9H), 0.18 (s,3H), 0.17 (s, 3H), 0.06 (s, 3H), 0.03 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ170.1, 138.3, 128.5, 127.7, 127.6, 97.2, 76.4, 74.9, 73.5, 69.1, 68.5,67.0, 25.9, 25.8, 21.7, 18.2, 18.1, −3.9, −4.1, −4.5, −5.0; FAB MS(C₂₇H₄₇N₃O₆Si₂) m/z (M⁺) calcd 565.3003, obsd 565.3011.

Synthesis of Glucosamine Donors6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosylfluoride 10α and6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosylfluoride 10β

tert-Butyldimethylsilyl6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside(4.0 g, 7.07 mmol) was dissolved in THF (60 mL) and cooled to 0° C.Glacial acetic acid (500 μL, 8.7 mmol) and TBAF (1 M in THF, 8.2 mL, 8.2mmol) were added simultaneously. After 30 min, the mixture was pouredinto ether (200 mL) and washed three times with brine. The organic layerwas dried over Na₂SO₄, filtered and the solvents were removed in vacuo.The residue was coevaporated with toluene, dissolved in anhydrous THF(20 mL) and cooled to −30° C. DAST (1.2 mL, 9.08 mmol) was addeddropwise and the mixture was stirred for 5 min at −30° C. and 30 min atroom temperature. The reaction mixture was cooled to −30° C. andanhydrous methanol (500 μL) was added. After warming to roomtemperature, the mixture was poured into EtOAc (300 mL) and washed withsaturated NaHCO₃, water and brine. The organic layer was dried overNa₂SO₄, filtered and the solvents were removed in vacuo. Flashchromatography on silica gel (Hexanes:EtOAc 95:5) afforded a mixture(5:1) of 10α and 10β (3.0 g, 6.62 mmol, 94%) as a crystalline solid.10α: ¹H-NMR (300 MHz, CDCl₃) δ 7.45-7.28 (m, 5H), 5.68 (dd, J=2.7, 52.7,1H), 4.92 (d, J=11.0 Hz, 1H), 4.84 (d, J=11.0 Hz, 1H), 4.45 (dd, J=1.9,12.1 Hz, 1H), 4.11 (dd, J=4.7 Hz, 12.1 Hz, 1H), 4.04-3.94 (m, 1H),3.83-3.71 (m, 2H), 3.74-3.36 (m, 1H), 2.11 (s, 3H), 0.92 (s, 9H), 0.06(s, 3H), 0.05 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 170.7, 137.7, 128.5,127.8, 127.5, 107.6, 104.6, 80.1, 75.6, 73.14, 73.08, 70.4, 64.3, 64.0,62.5, 26.1, 21.1, 18.3, −3.4, −4.7. 10β: ¹H-NMR (300 MHz, CDCl₃) δ7.45-7.28 (m, 5H), 5.68 (dd, J=2.7 Hz, 52.7 Hz, 1H), 4.92 (d, J=11.0 Hz,1H), 4.84 (d, J=11.0 Hz, 1H), 4.45 (dd, J=1.9 Hz, 12.1 Hz, 1H), 4.11(dd, J=4.7 Hz, 12.1 Hz, 1H), 4.04-3.94 (m, 1H), 3.83-3.71 (m, 2H),3.74-3.36 (m, 1H), 2.11 (s, 3H), 0.92 (s, 9H), 0.06 (s, 3H), 0.05 (s,3H); ¹³C-NMR (75 MHz, CDCl₃) δ 170.7, 137.7, 128.6, 128.5, 128.2, 127.9,127.6, 109.6, 106.7, 82.4, 82.3, 75.4, 74.8, 74.7, 70.2, 66.6, 66.3,62.8, 26.0, 21.1, 18.2, −3.5, −4.7.

O-(6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)trichloroacetimidate 1160 andO-(6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyl)trichloroacetimidate 11β

tert-Butyldimethylsilyl6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside(1.16 g, 2.05 mmol) was dissolved in anhydrous THF (20 mL) and cooled to0° C. Glacial acetic acid (146 μl, 2.56 mmol) and TBAF (1M in THF) (2.25mL, 2.25 mmol) were added simultaneously. After 30 min, the mixture waspoured into ether (200 mL) and washed three times with brine. Theorganic layer was dried over Na₂SO₄, filtered and the solvents wereremoved in vacuo. The residue was dissolved in CH₂Cl₂ (50 mL) and cooledto 0° C. Trichloroacetonitrile (3.1 mL, 30.9 mmol) and DBU (30 μL, 0.2mmol) were added and the mixture was stirred for 1 h at 0° C. andconcentrated in vacuo. Flash chromatography on silica gel (Hexanes:EtOAc85:15) afforded a mixture of 11α and 11β (27/73) (1.12 g, 1.88 mmol,92%) as a colorless oil. 11α: [α²⁴ _(D): +118.6 (c 1.69, CH₂Cl₂); IR(thin film) 3344, 2954, 2929, 2857, 2110, 1745, 1674, 1363, 1255, 1142,1069, 1021, 836 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 8.75 (s, 1H, NH),7.41-7.28 (m, 5H, arom. H), 6.44 (d, J=3.4 Hz, 1H, H-1), 4.92 (d, J=11.3Hz, 1H, benzyl-CH₂), 4.86 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.40 (dd,J=12.2, 2.1 Hz, 1H, H-6a), 4.08 (dd, J=12.2, 4.6 Hz, 1H, H-6b),3.95-3.99 (m, 1H), 3.77-3.82 (m, 2H), 3.66-3.71 (m, 1H), 2.06 (s, 3H,acetyl-CH₃), 0.91 (s, 9H, tert-butyl), 0.06 (s, 3H, CH₃), 0.05 (s, 3H,CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 170.8, 160.9, 137.9, 128.5, 127.8,127.6, 94.8, 80.5, 75.5, 73.3, 70.8, 63.7, 62.7, 26.1, 21.0, 18.2, −3.5,−4.8; FAB MS (C₂₃H₃₃Cl₃N₄O₆Si) m/z (M⁺) calcd 594.1235, obsd 594.1219.11β: [α]²⁴ _(D): +43.6 (c 1.11, CH₂Cl₂); IR (thin film) 3329, 2928,2857, 2113, 1745, 1676, 1253, 1098, 1063, 838 cm⁻¹; ¹H-NMR (500 MHz,CDCl₃) δ 8.74 (s, 1H, NH), 7.48-7.28 (m, 5H, arom. H), 5.66 (d, J=8.2Hz, 1H, H-1), 4.94 (d, J=11.3 Hz, 1H, benzyl-CH₂), 4.79 (d, J=11.3 Hz,1H, benzyl-CH₂), 4.42 (dd, J=11.9, 2.4 Hz, 1H, H-6a), 4.13 (dd, J=12.2,4.9 Hz, 1H, H-6b), 3.77 (dd, J=9.5, 8.5 Hz, 1H), 3.69 (dd, J=9.8, 8.2Hz, 1H), 3.60 (ddd, J=9.5, 4.9, 2.4 Hz, 1H, H-5), 3.36 (dd, J=9.5, 8.5Hz, 1H), 2.08 (s, 3H, acetyl-CH₃), 0.90 (s, 9H, tert-butyl), 0.05 (s,3H, CH₃), 0.04 (s, 3H, CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 170.9, 161.2,138.1, 128.6, 127.9, 127.6, 97.1, 83.4, 75.5, 75.4, 70.4, 66.2, 62.8,26.0, 21.1, 18.2, −3.5, −4.8; FAB MS (C₂₃H₃₃Cl₃N₄O₆Si) m/z (M⁺) calcd594.1235, obsd 594.1222.

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosylbromide 12

TBAF (1.0 M in THF, 6.4 mL) and glacial acetic acid (350 μL, 5.9 mmol)were added dropwise to a solution of tert-butyldimethylsilyl3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside(2.90 g, 5.6 mmol) in THF (60 mL) under nitrogen at 0° C. The reactionmixture was warmed to room temperature, stirred for 1.5 h and quenchedwith saturated NaHCO₃. After extracting with CH₂Cl₂ (3×), the combinedorganic phases were dried over MgSO₄, filtered and the solvents wereremoved in vacuo. The crude material was evaporated three times withtoluene, dried under vacuum for 1 h and dissolved in THF (17 mL). Theresulting solution was added to a suspension of SOBr₂ (760 μL, 9.6 mmol)and imidazole (585 mg, 8.6 mmol) in anhydrous THF (55 mL) at 0° C. Theresulting suspension was stirred for 1 h, diluted with anhydrous ether,filtered over a pad of florisil and ground Na₂S₂O₃ and concentrated tofurnish 12 as a yellow solid (2.1 g, 4.5 mmol, 76%) which was usedwithout further purification. [α]²⁴ _(D): +5.8 (c 1.00, CHCl₃); IR (thinfilm) 2929, 2859, 2113, 1746, 1473, 1235, 1006 cm⁻¹; ¹H-NMR (500 MHz,CDCl₃) δ 6.40 (d, J=3.9 Hz, 1H, H-1), 5.43 (dd, J=8.8, 10.4 Hz, 1H,H-3), 4.41 (dd, J=2.1, 12.5 Hz, 1H, H-6a), 4.17-4.11 (m, 2H, H-6b, H-5),3.87 (t, J=8.8 Hz, 1H, H-4), 3.58 (dd, J=3.9, 10.4 Hz, 1H, H-2), 2.17(s, 3H, OCH₃), 2.10 (s, 3H, OCH₃), 0.86 (s, 9H, C(CH₃)₃), 0.07 (s, 3H,SiCH₃), 0.06 (s, 3H, SiCH₃); ¹³C-NMR (100 MHz, CDCl₃) δ 170.6, 169.6,88.1, 75.1, 73.7, 68.6, 63.4, 61.9, 25.8, 21.5, 20.9, 18.1, −3.8, −4.8;FAB MS (C₁₆H₂₈BrN₃O₇Si) m/z (M⁺) calcd 465.0930, obsd 465.0940.(Procedure: D. K. Baeschlin, A. R. Chaperon, L. G. Green, M. G. Hahn, S.J. Ince, S. V. Ley, Chem. Eur. J. 2000, 6, 172-186).

O-(3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)trichloroacetimidate 13α andO-(3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethyl-silyl-2-deoxy-β-D-glucopyranosyl)trichloroacetimidate 13β

TBAF (1.0 M in THF, 1.4 mL) and glacial acetic acid (80 μL, 5.9 mmol)were added dropwise to a solution of tert-butyldimethylsilyl3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside(590 mg, 1.14 mmol) in THF (12 mL) under nitrogen at 0° C. The reactionmixture was warmed to room temperature, stirred for 1.5 h and quenchedwith saturated NaHCO₃ solution. After extraction with CH₂Cl₂ (3×), thecombined organic phases were dried over MgSO₄, filtered and the solventswere removed in vacuo. The crude material was dried by coevaporationwith anhydrous toluene and vacuum for 1 h and dissolved in CH₂Cl₂ (25mL). Trichloroacetonitrile (1.3 mL, 12.50 mmol) and freshly activated 4Å powdered molecular sieves (300 mg) were added and the mixture wasstirred for 30 minutes at room temperature. After cooling to 0° C., DBU(30 μl, 0.2 mmol) was added and the temperature was allowed to rise toroom temperature. After 1h, the mixture was filtered through Celite andthe solvents were removed in vacuo. Flash chromatography on silica gel(Hexanes:EtOAc 85:15) afforded 13α (437 mg, 0.80 mmol, 70%) and 13β (94mg, 0.17 mmol, 15%). 13α: ¹H-NMR (500 MHz, CDCl₃) δ 8.81 (s, 1H, NH),6.44 (d, J=3.6 Hz, 1H, H-1), 5.43 (dd, J=8.8, 10.4 Hz, 1H, H-3), 4.41(dd, J=1.9, 12.0 Hz, 1H, H-6a), 4.12-3.99 (m, 2H, H-6b, H-5), 3.87 (t,J=9.1 Hz, 1H, H-4), 3.58 (dd, J=3.6, 10.7 Hz, 1H, H-2), 2.17 (s, 3H),OCH₃), 2.01 (s, 3H, OCH₃), 0.86 (s, 9H, C(CH₃)₃), 0.08 (s, 3H, SiCH₃),0.06 (s, 3H, SiCH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 170.5, 169.7, 160.8,94.7, 72.77, 69.1, 62.5, 61.5, 25.9, 21.6, 21.0, 18.2, −3.8, −4.6. 13β:¹H-NMR (500 MHz, CDCl₃) δ 8.81 (s, 1H, NH), 5.73 (d, J=8.2 Hz, 1H, H-1),5.03 (dd, J=8.8 Hz, J=9.8 Hz, 1H, H-3), 4.40 (dd, J=2.1 Hz, J=11.9 Hz,1H, H-6a), 4.13 (dd, J=4.2 Hz, J=11.9 Hz, 1H, H-6b), 3.87 (t, J=9.5 Hz,1H, H-4), 3.58 (m, 2H, H-2, H-5), 2.17 (s, 3H, OCH₃), 2.08 (s, 3H,OCH₃), 0.84 (s, 9H, C(CH₃)₃), 0.05 (s, 3H, SiCH3), 0.04 (s, 3H, SiCH₃);¹³C-NMR (125 MHz, CDCl₃) δ 170.7, 169.9, 160.8, 96.6, 75.3, 68.8, 64.1,62.4, 25.9, 25.8, 25.8, 21.5, 21.1, 18.1, −3.9, −4.8.

O-(3,6-Di-O-benzyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-D-glucopyranosyl)trichloroacetimidate 14

tert-Butyldimethylsilyl6-O-benzyl-2-azido-3-O-acetyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside8 (0.121 mg, 0.197 mmol) was dissolved in anhydrous THF (1.5 mL) andcooled to 0° C. Glacial acetic acid (20.0 μL, 0.256 mmol) and TBAF (1Min THF, 240 μl, 0.240 mmol) were added simultaneously. After 30 min, themixture was poured into EtOAc (50 mL) and washed with sat. NaHCO₃. Theorganic layer was dried over Na₂SO₄, filtered and the solvents wereremoved in vacuo. The residue was dissolved in CH₂Cl₂ (1 mL) and cooledto 0° C. Trichloroacetonitrile (1.0 mL) and DBU (5 μL, 0.03 mmol) wereadded and the mixture was stirred for 1 h at 0° C., diluted with CH₂Cl₂(30 mL), passed through a plug of silica and concentrated in vacuo.Flash chromatography on silica gel (Hexanes:EtOAc 5:1) afforded 14α and14β (87.5 mg, 0.137 mmol, 69%) as an inseparable 1:1 mixture. ¹H-NMR(500 MHz, CDCl₃) δ 8.76 (s, 1H, NHβ), 8.73 (s, 1H, NHα), 7.40-7.27 (m,15H, arom. H), 6.48 (d, J=3.6 Hz, 1H, H1α), 8.24 (d, J=8.2, 1H, H1β),4.94 (d, J=11.6 Hz, 1H), 4.91 (d, J=11.9 Hz, 1H), 4.85 (d, J=11.0 Hz,1H), 4.78 (d, J=11.3 Hz, 1H), 4.66 (d, J=12.2 Hz, 1H), 4.60 (d, J=12.2Hz, 1H), 4.53 (d, J=12.5 Hz, 1H), 4.50 (d, J=11.9 Hz, 1H), 3.95-3.90 (m,1H), 3.86 (app t, J=9.0 Hz, 1H), 3.81-3.73 (m, 3H), 3.71-3.64 (m, 5H),3.60-3.57 (m, 2H), 3.38 (app t, J=9.0 Hz, 1H), 0.88 (s, 9H), 0.87 (s,9H), 0.06-0.02 (m, 12H). ¹³C-NMR (125 MHz, CDCl₃) δ 161.2, 161.0 138.4,138.3, 138.1, 128.5, 128.5, 128.5, 127.8, 127.7, 127.6, 97.1, 95.2,83.5, 80.6, 77.7, 75.4, 75.2, 73.3, 70.6, 70.4, 68.4, 66.3, 63.9, 26.2,26.1, 18.2, 18.2, −3.5, −3.6, −4.6.

O-(6-O-Benzyl-2-azido-3-O-acetyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyl)trichloroacetimidate 15α andO-(6-O-Benzyl-2-azido-3-O-acetyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyl)trichloroacetimidate 15β

tert-Butyldimethylsilyl6-O-benzyl-2-azido-3-O-acetyl-4-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranoside9 (0.170 mg, 0.30 mmol) was dissolved in anhydrous THF (3 mL) and cooledto 0° C. Glacial acetic acid (20 μL, 0.35 mmol) and TBAF (1M in THF)(330 μL, 0.33 mmol) were added simultaneously. After 30 min, the mixturewas poured into ether (50 mL) and washed three times with brine. Theorganic layer was dried over Na₂SO₄, filtered and the solvents wereremoved in vacuo. The residue was dissolved in CH₂Cl₂ (3 mL) and cooledto 0° C. Trichloroacetonitrile (770 μl, 7.67 mmol) and DBU (5 μL, 0.03mmol) were added and the mixture was stirred for 1 h at 0° C. andconcentrated in vacuo. Flash chromatography on silica gel (Hexanes:EtOAc85:15) afforded a mixture of 15α and 15β (2.7/1) (0.160 mg, 0.27 mmol,89%) as a colorless oil. 15α ¹H-NMR (500 MHz, CDCl₃) δ 8.77 (s, 1H, NH),7.36-7.27 (m, 5H, arom. H), 6.50 (d, J=3.4 Hz, 1H, H-1), 5.43 (dd,J=7.9, 10.7 Hz, 1H, H-3), 4.58 (d, J=11.9 Hz, 1H, benzyl-CH₂), 4.51 (d,J=11.9 Hz, 1H, benzyl-CH₂), 4.01-3.95 (m, 2H, H-4, H-5), 3.75 (dd,J=3.3, 11.3 Hz, 1H, H-6a), 3.65 (dd, 1H, H-6b), 3.48 (dd, J=3.4, 10.4Hz, 1H, H-2), 2.17 (s, 3H, acetyl-CH₃), 0.84 (s, 9H, tert-butyl), 0.07(s, 6H, CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 170.0, 161.0, 138.1, 128.5,127.8, 127.7, 95.2, 74.7, 73.6, 73.1, 68.7, 68.0, 61.7, 25.9, 21.6,18.2, −4.0, −4.6. 15β: [α]²⁴ _(D): +x (c x, CH₂Cl₂); IR (thin film) XXcm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 8.78 (s, 1H, NH), 7.34-7.27 (m, 5H,arom. H), 5.76 (d, J=8.5 Hz, 1H, H-1), 5.03 (dd, J=8.8, 10.0 Hz, 1H,H-3), 4.64 (d, J=12.2 Hz, 1H, benzyl-CH₂), 4.53 (d, J=12.3 Hz, 1H,benzyl-CH₂), 3.91 (t, J=9.1 Hz, 1H, H-4), 3.68-3.58 (m, 4H, H-2, H-5,H6a, H6b), 2.16 (s, 3H, acetyl-CH₃), 0.82 (s, 9H, tert-butyl), 0.06 (s,3H, CH₃), 0.04 (s, 3H, CH₃).

Synthesis of Non-Reducing End Monosaccharidetert-Butyldimethylsilyl-6-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside16

tert-Butyldimethylsilyl3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside 2 (1.23 g; 2.77mmol) was dissolved in methanol (10 mL). NaOMe (25% in methanol, 170 μL)was added. After 15 min DOWEX-50 acidic resin was added and the mixturewas stirred until the pH reached 6. The DOWEX resin was filtered off andthe solvent was removed under reduced pressure to afford a yellow oil.The residue was coevaporated twice with toluene, dissolved in2,4,6-collidine (7 mL), cooled to −40° C. and acetyl chloride (196 μL,2.74 mmol) was added. After stirring the reaction mixture for 3 h, asecond portion of acetyl chloride (42 μL, 0.6 mmol) was added. Themixture was stirred for another 1 h at −40° C. and for 1 h at roomtemperature and then quenched with saturated NaHCO₃. After addition ofCH₂Cl₂ and phase separation the aqueous phase was extracted three timeswith CH₂Cl₂. The combined organic phases were dried over MgSO₄, filteredand the solvents were removed in vacuo. Flash chromatography(Hexanes:EtOAc 4:1) on silica afforded 16 (933 mg, 2.58 mmol, 93%) as acolorless syrup. [α]²⁴ _(D): −7.0 (c 1.00, CHCl₃); IR (thin film) 3412,2929, 2958, 2111, 1741, 1463, 1370, 1257, 1177 cm⁻¹; ¹H-NMR (500 MHz,CDCl₃) δ 4.55 (d, J=7.5 Hz, 1H), 4.38-4.26 (m, 2H), 4.13 (bs, 2H, OH),3.45-3.20 (m, 4H), 2.06 (s, 3H, OCH₃), 0.93 (s, 9H, C(CH₃)₃), 0.15 (s,3H, SiCH₃), 0.14 (s, 3H, SiCH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 6 171.9,97.3, 74.5, 73.8, 70.6, 68.2, 63.7, 25.7, 21.0, 18.1, −3.5, −4.6; FAB MS(C₁₄H₂₇N₃O₆Si) m/z (M⁺) calcd 361.1669, obsd 361.1680.

tert-Butyldimethylsilyl-6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranoside17

tert-Butyldimethylsilyl-6-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside16 (7.1 g, 19.64 mmol) was dissolved in CH₂Cl₂ (100 mL). Powdered,freshly activated 4 Å molecular sieves (20 g) and benzyl bromide (12 mL,100 mmol) were added and this mixture stirred for 30 min. Silver(I)oxide(26.4 g, 114 mmol) was added and light was excluded from the reactionmixture. After 48 h, the reaction mixture was filtered over Celite andthe filtrate was concentrated under reduced pressure. Flashchromatography on silica gel (Hexanes-EtOAc 97:3) afforded 17 (8.5 g,15.7 mmol, 80%) as a colorless oil. [α]²⁴ _(D): −4.7 (c 1.40, CH₂Cl₂);IR (thin film) 3031, 2955, 2858, 2109, 1745, 1454, 1252, 1042, cm⁻¹;¹H-NMR (500 MHz, CDCl₃) δ 7.43-7.29 (m, 10H, arom. H), 4.95 (d, J=11.0Hz, 1H, benzyl-CH₂), 4.89 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.82 (d,J=10.7 Hz, 1H, benzyl-CH₂), 4.61 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.56(d, J=7.6 Hz, 1H, H-1), 4.36 (dd, J=11.9,1.5 Hz, 1H, H-6a), 4.17 (dd,J=11.9, 5.8 Hz, 1H, H-6b), 3.55-3.49 (m, 2H, H-4 and H-5), 3.47-3.43 (m,1H, H-3), 3.38 (dd, J=9.8, 7.6 Hz, 1H, H-2), 2.06 (s, 3H, acetyl-CH₃),0.98 (s, 9H, tert-butyl), 0.20 (s, 3H, CH₃), 0.19 (s, 3H, CH₃); ¹³C-NMR(125 MHz, CDCl₃) δ 170.8, 138.0, 137.7, 128.7, 128.6, 128.2, 128.2,128.1, 97.3, 83.1, 77.7, 75.7, 75.2, 73.2, 68.8, 63.2, 25.8, 21.0, 18.2,−4.2, −5.1; FAB MS (C₂₈H₃₉N₃O₆Si) m/z (M⁺) calcd 541.2608, obsd541.2606.

O-(6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)trichloroacetimidate 18α andO-(6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranosyl)trichloroacetimidate 18β

To a solution of 17 (756 mg, 1.4 mmol) in anhydrous THF (15 mL) at 0° C.glacial acetic acid (100 μl, 1.75 mmol) and TBAF (1 M in THF, 1.55 mL,1.55 mmol) were added simultaneously. After 30 min this mixture waspoured into ether (150 mL) and extracted three times with brine. Theorganic layer was dried over Na₂SO₄, filtered and the solvents wereremoved in vacuo. The residue was dissolved in anhydrous dichloromethane(50 mL) and cooled in an ice bath. Trichloroacetonitrile (2.1 mL, 21mmol) and DBU (21 μL, 0.14 mmol) were added. After 45 min, the solventswere removed in vacuo. Flash chromatography on silica gel (Hexanes:EtOAc85:15→8:2) afforded a mixture (58:42) of 18α and 18β (708 mg, 1.24 mmol,88%) as a colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 8.76 (s, 1H, NH),7.44-7.26 (m, 10H, arom. H), 6.42 (d, J=3.4 Hz, 0.58H, H-1α), 5.64 (d,J=8.2 Hz, 0.42H, H-1β), 4.97-4.86 (m, 4H, benzyl-CH₂), 4.64-4.60 (m,1H), 4.35-4.24 (m, 2H), 4.10-4.06 (m, 1H), 3.73-3.57 (m, 2H), 2.03 (s,3H, acetyl-CH₃).

Synthesis of Uronic Acid Monosaccharides Methyl3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranosiduronate 20

1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose 19 (52.06 g, 200 mmol) wasdissolved in THF (500 mL) and NaH (60% in mineral oil, washed withpentanes) (9.6 g, 240 mmol) was added in portions. After the evolutionof hydrogen ceased, tetrabutylammonium iodide (500 mg, 1.35 mmol) andbenzyl bromide (25 ml, 210 mmol) were added and the mixture stirred for10 h at room temperature. Water was added slowly to the reaction mixtureand the organic solvents were removed in vacuo. The aqueous phase wasextracted three times with EtOAc. The combined organic phases were driedover Na₂SO₄, filtered through a plug of silica gel and the solvents wereremoved in vacuo.

Aqueous acetic acid (66%, 300 mL) was added to the resulting oil and themixture was stirred for 14 h at room temperature and for 6 h at 40° C.After removal of the solvents, the remaining residue was dissolved inCH₂Cl₂ and extracted with saturated NaHCO₃. After phase separation, theaqueous layer was extracted with CH₂Cl₂. The combined organic phaseswere dried over Na₂SO₄, filtered and the solvents were removed in vacuo.

The residual oil was dissolved in CH₂Cl₂ (750 mL) and pyridine (80 mL),before DMAP (3,5 g, 28.6 mmol) and tert-butyldimethylsilyl chloride (32g, 212 mmol) were added. After stirring at room temperature for 19 h,the mixture was extracted with water, 1 N HCl, brine and saturatedNaHCO₃. The organics were dried over Na₂SO₄, filtered and the solventswere removed in vacuo. The residue was dissolved in anhydrous pyridine(170 mL) and DMAP (1 g, 8.2 mmol) was added. The mixture was cooled to0° C. and acetic anhydride (38 ml, 403 mmol) was added dropwise. Afterstirring overnight at room temperature, the solvents were removed invacuo. The residue was dissolved in EtOAc and extracted with water, 1 NHCl, brine and saturated NaHCO₃. The organic layer was dried overNa₂SO₄, filtered and the solvents were removed in vacuo.

The residue (91.2 g, max. 195 mmol) was dissolved in THF (300 mL) andcooled to 0° C. To this solution, HF-pyridine (24 mL) in pyridine (80mL) was added and stirred overnight at room temperature. The reactionmixture was poured into water and extracted three times with EtOAc. Thecombined organic phases were extracted with water, 1 N HCl, water,saturated NaHCO₃, dried over Na₂SO₄, filtered and the solvents wereremoved in vacuo.

The residue was dissolved in CH₂Cl₂ (400 mL) and TEMPO (800 mg, 5.1mmol) was added. A mixture of saturated NaHCO₃ (700 mL), water (200 mL),KBr (2.25 g, 18.9 mmol) and tetrabutylammonium bromide (3.45 g, 10.7mmol) was added and the resulting mixture was cooled to 0° C. Withvigorous stirring, commercially available household bleach (700 mL) wasadded in 100 mL portions every 10 min. After complete addition, stirringwas continued for 30 min and methanol was added until the mixture wasdecolorized. After 20 min, the aqueous layer was extracted with CH₂Cl₂followed by the dropwise addition of conc. HCl to pH 1. The aqueouslayer was extracted three times with CH₂Cl₂. The organic layers weredried over Na₂SO₄, filtered and the solvents were removed in vacuo.

The residue was dissolved in methanol (280 mL), 4 N NaOH (42 mL) wasadded and the mixture was stirred overnight at room temperature. Themixture was acidified with conc. HCl and extracted five times withCH₂Cl₂. The combined organic phases were dried over Na₂SO₄, filtered andthe solvents were removed in vacuo.

The residue was dissolved in anhydrous DMF (200 mL) and powdered KHCO₃(27.4 g, 274 mmol) and methyl iodide (17 mL, 273 mmol) were added. Afterstirring at room temperature overnight, the mixture was poured intoether and extracted twice with water, saturated Na₂SO₃, and water. Theorganic phase was dried over Na₂SO₄, filtered and the solvents wereremoved in vacuo to afford 20 (44 g, 130 mmol, 65%) as a slightly yellowoil. ¹H-NMR (300 MHz, CDCl₃) δ 7.40-7.27 (m, 5H), 6.04 (d, J=3.9 Hz,1H), 4.70-4.52 (m, 4H), 4.41 (dd, J=6.2, 3.8 Hz, 1H), 4.16 (d, J=3.8 Hz,1H), 3.76 (s, 3H), 3.34 (d, J=9.1 Hz, 1H), 1.50 (s, 3H), 1.34 (s, 3H).The spectral data was in agreement with the reported data (J.-C.Jacquinet, M. Petitou, P. Duchaussoy, I. Lederman, J. Choay, G. Torri,P. Sinay. Carbohydrate Research 1984, 130, 221-241).

Methyl 3-O-benzyl-D-glucopyranosiduronate 21

Methyl 3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranosiduronate 20(3.43 g, 10.14 mmol) was dissolved in 90% aqueous trifluoroacetic acid(20 mL) and stirred for 15 min at room temperature. The solvent wasevaporated and the residue coevaporated twice with water and twice withtoluene to afford methyl 3-O-benzyl-D-glucopyranosiduronate 21, whichwas used without further purification. The spectral data was inagreement with the reported data (J.-C. Jacquinet, M. Petitou, P.Duchaussoy, I. Lederman, J. Choay, G. Torri, P. Sinay. CarbohydrateResearch 1984, 130, 221-241).

Methyl3-O-benzyl-1,2-O-isopropylidene-5-O-levulinoyl-β-L-idofuranosiduronate22

A solution of trifluoromethanesulfonic anhydride (13 mL) in CH₂Cl₂ (250mL) was added dropwise to a mixture of pyridine (13 mL) and CH₂Cl₂ (132mL) at −20° C. The mixture was allowed to warm to −10° C. and a solutionof methyl 3-O-benzyl-1,2-O-isopropylidene-α-D--glucofuranosiduronate 20(12 g, 35.5 mmol) in CH₂Cl₂ (123 mL) was added dropwise. After 1 h at−10° C., the mixture was poured into ice cold water containing NaHCO₃and stirred for 1 h. The organic layer was washed with 3% HCl, water,dried over MgSO₄, filtered and the solvents were removed in vacuo.Sodium levulinate (9.8 g, 71 mmol) was added to a solution of the cruderesidue in DMF (65 mL) and the resulting mixture was stirred overnightat 80° C. and cooled to room temperature. After dilution with EtOAc, themixture was washed with water and the organic phase was dried overMgSO₄, filtered and the solvents were removed in vacuo. Flashchromatography on silica gel (Toluene:EtOAc 9:1→7:3) afforded 22 (12.8g, 29.3 mmol, 82%) as an amorphous solid. [α]²⁴ _(D): −4.2 (c 1, CHCl₃);IR (thin film) 2512, 1751, 1718, 1025 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ7.36-7.28 (m, 5H, arom. H), 5.97 (d, J=3.9 Hz, 1H, H-1), 5.53 (d, J=7.0Hz, 1H, H-5), 4.66-4.62 (m, 3H, benzyl-CH_(2a), H-2, H-4), 4.50 (d,J=11.4 Hz, 1H, benzyl-CH_(2b)), 4.16 (d, 1H, H-3), 3.69 (s, 3H, OCH₃),2.73-2.63 (m, 4H, levulinic-CH₂), 2.12 (s, 3H, OCH₃), 1.59 (s, 3H, CH₃),1.34 (s, 3H, CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 205.3, 171.9, 168.5,137.2, 128.6, 128.4, 128.0, 112.7, 105.1, 83.1, 82.7, 72.5, 70.9, 52.8,38.0, 30.0, 28.0, 27.3, 26.8; FAB MS (C₂₂H₂₈O₉) m/z (M⁺) calcd 436.1733,obsd 436.1742.

Methyl 3-O-benzyl-L-idopyranosiduronate 23

Hydrazine hydrate (7.3 mL, 146 mmol) was added to a solution of methyl3-O-benzyl-1,2-O-isopropylidene-5-O-levulinoyl-β-L-idofuranosiduronate22 (12.8 g, 29.3 mmol) in pyridine-acetic acid (3:2, 290 mL) at 0° C.After 15 min, acetone (1.2 L) was added, and the mixture was stirred atroom temperature for 15 min. After removal of the solvents in vacuo, thecrude product was dissolved in aqueous trifluoroacetic acid (90%, 70mL). The mixture was stirred for 15 min, the solvents removed in vacuoand coevaporated twice with water to give a white solid, which wasrecrystallized from ethyl acetate/hexanes to afford 23 (8.0 g, 26.8mmol, 91%). Analytical data was in agreement with reported data (J.-C.Jacquinet, M. Petitou, P. Duchaussoy, I. Lederman, J. Choay, G. Torri,P. Sinay. Carbohydrate Research 1984, 130, 221-241).

Methyl 3-O-benzyl-1,2-O-isopropylidene-α-D-glucopyranosiduronate 24

Methyl 3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranosiduronate 21(3.43 g, 10.14 mmol) was dissolved in trifluoroacetic acid (90% aqueous,20 mL) and stirred for 15 min at room temperature. The solvent wasremoved under reduced pressure and the residue coevaporated twice withwater and twice with toluene. The residue was dissolved in DMF (10 mL)and 2-methoxypropene (10 mL, 100 mmol) and cooled to 0° C. A solution of(1S)-(+)-camphorsulfonic acid (230 mg, 1 mmol) in DMF (2 mL) was addedand stirring was continued at 0° C. for 1 h and at room temperatureovernight. Methanol (15 mL) was added and the mixture was stirred for 3h at room temperature. Triethylamine (3 mL) was added and the mixturewas concentrated. The residue was dissolved in Et₂O and washed withwater and twice with brine. The organic layer was dried over Na₂SO₄ andafter filtration the solvent was removed under reduced pressure. Flashchromatography on silica gel (hexanes:EtOAc 9:1) afforded methyl3-O-benzyl-1,2-O-isopropylidene-α-D-glucopyranosiduronate 24 (1.65 g,4.88 mmol, 48%) as a colorless oil. [α]²⁴ _(D): +22.3 (c 1.13, CH₂Cl₂);IR (thin film on NaCl) 3520, 3037, 2987, 1752, 1455, 1169, 1105 cm⁻¹;¹H-NMR (500 MHz, CDCl₃) δ 7.34-7.26 (m, 5H, arom. H), 5.87 (d, J=2.7 Hz,1H, H-1), 4.63 (d, J=11.9 Hz, 1H, benzyl-CH₂), 4.54 (d, J=11.6 Hz, 1H,benzyl-CH₂), 4.54 (d, J=3.7 Hz, 1H, H-5), 4.24-4.20 (m, 1H, H-4),4.11-4.09 (m, 1H, H-3), 4.00 (dd, J=3.1 Hz, J=3.4 Hz, 1H, H-2), 3.58 (s,3H, OCH₃), 3.51 (d, J=10.4 Hz, 1H, OH), 1.60 (s, 3H, CH₃), 1.37 (s, 3H,CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 170.3, 137.2, 128.6, 128.2, 127.8,111.4, 94.4, 75.8, 75.5, 73.7, 72.4, 67.0, 52.2, 28.0, 25.9; FAB MS(C₁₇H₂₂O₇) m/z (M)⁺ calcd 338.1366, obsd 338.1377. Further elution(hexanes-EtOAc 7:3) afforded 25 (1.1 g, 32%) as a colorless oil.

Methyl 3-O-benzyl-1,2-O-cyclopentylidene-α-D-glucopyranosiduronate 26and Methyl 3-O-benzyl-1,2-O-cyclopentylidene-α-D-glucofuranosiduronate27

Methyl 3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranosiduronate 21(3.34 g, 9.87 mmol) was dissolved in trifluoroacetic acid (90% aqueous,20 ml) and stirred for 15 min at room temperature. The solvent wasremoved under reduced pressure and the residue coevaporated twice withwater and twice with toluene. The residue was dissolved in DMF (10 mL)and methoxycyclopentene (7.1 g, 72 mmol) and cooled to 0° C. A solutionof (1S)-(+)-camphorsulfonic acid (244 mg, 1.05 mmol) in DMF (2 mL) wasadded and stirring was continued at 0° C. for 1 h and at roomtemperature overnight. Methanol (5 mL) was added and the mixture wasstirred for 30 min at room temperature. Triethylamine (3 mL) was addedand the mixture was concentrated. The residue was dissolved in Et₂O andwashed with water and brine (2×). The organic layer was dried overNa₂SO₄ and after filtration the solvent was removed under reducedpressure. Flash chromatography on silica gel (toluene-EtOAc 98.5:1.5)afforded methyl3-O-benzyl-1,2-O-cyclopentylidene-α-D-glucopyranosiduronate 26 (2.05 g,57%) as a colourless oil. [α]²⁴ _(D): +33.9 (c 1.48, CH₂Cl₂); IR (thinfilm on NaCl) 3510, 3032, 2955, 2873, 1750, 1454, 1436, 1336, 1206 cm⁻¹;¹H-NMR (500 MHz, CDCl₃) δ 7.37-7.27 (m, 5H, arom.), 5.84 (d, J=2.8 Hz,1H, H-1), 4.66 (d, J=11.6 Hz, 1H, benzyl-CH₂), 4.57 (d, J=11.9 Hz, 1H,benzyl-CH₂), 4.52 (d, J=4.3 Hz, 1H, H-5), 4.23-4.19 (m, 1H, H-4), 4.04(dd, J=3.0, 2.4 Hz, 1H, H-2), 4.00 (dd, J=3.4,3.1 Hz, 1H, H-3), 3.63 (s,3H, OCH₃), 3.40 (d, J=9.5 Hz, 1H, OH), 2.14-2.08 (m, 1H, cyclopentenyl),1.92-1.86 (m, 1H, cyclopentenyl), 1.79-1.62 (m, 6H, cyclopentenyl);¹³C-NMR (125 MHz, CDCl₃) δ 170.5, 137.3, 128.6, 128.2, 127.8, 94.2,75.9, 75.0, 74.2, 72.4, 67.4, 52.3, 37.7, 36.8, 23.5, 23.2; FAB MS(C₁₉H₂₄O₇) m/z (M)⁺ calcd 364.1522, obsd 364.1534. Further elution(toluene:EtOAc 9:1) afforded methyl3-O-benzyl-1,2-O-cyclopentylidene-α-D-glucofuranosiduronate 27 (1.04 g,2.85 mmol, 29%) as a colorless oil. [α]²⁴ _(D): +0.7 (c 1.37, CH₂Cl₂);IR (thin film on NaCl) 3470, 2955, 2875, 1738, 1455, 1208 cm⁻¹; ¹H-NMR(400 MHz, CDCl₃) δ 7.37-7.27 (m, 5H, arom.), 5.98 (d, J=4.0 Hz, 1H,H-1), 4.66 (d, J=11.5 Hz, 1H, benzyl-CH₂), 4.63-4.52 (m, 3H, benzyl-CH₂,H-3, H-5), 4.43 (dd, J=6.0 Hz, J=4.0 Hz, 1H, H-4), 4.16 (d, J=4.0 Hz,1H, H-2), 3.73 (s, 3H, OCH₃), 3.36 (br. s, 1H, OH), 2.00-1.93 (m, 1H,cyclopentenyl), 1.86-1.76 (m, 1H, cyclopentenyl), 1.74-1.60 (m, 6H,cyclopentenyl); ³C-NMR (100 MHz, CDCl₃) δ 173.1, 136.9, 128.7, 128.3,128.1, 121.9, 105.3, 83.3, 82.7, 80.1, 72.8, 70.1, 52.5, 37.1, 36.7,23.4, 23.2; FAB MS (C₁₉H₂₄O₇) m/z (M)⁺ calcd 364.1522, obsd 364.1530.

Methyl 3-O-benzyl-1,2-O-isopropylidene-β-L-idopyranosiduronate 28

Methyl 3-O-benzyl-L-idopyranosiduronate 23 (1.7 g, 5.70 mmol) wasdissolved in DMF (6 mL) and 2-methoxypropene (10.7 mL, 114 mmol) wasadded. The mixture was cooled at 0° C. and (1S)-(+)-camphorsulfonic acid(132 mg, 0.57 mmol) in DMF (2 mL) was added dropwise under stirring. Themixture was stirred for 6 h at 0° C., then methanol (2 mL) was added andstirred for 30 min at 0° C. before the reaction was quenched by additionof triethylamine After dilution with EtOAc, the solution was washed withwater. The organic layer was dried over MgSO₄ and evaporated. Flashchromatography on silica gel (toluene:EtOAc 99:1→96:4) afforded 28 (1.3g, 3.84 mmol, 68%) as a yellow oil. [α]²⁴ _(D): −29.5 (c 1.00, CHCl₃);IR (thin film on NaCl) 2937, 1764, 1374, 843 cm⁻¹; ¹H-NMR (500 MHz,CDCl₃) δ 7.40-7.30 (m, 5H, arom.), 5.35 (d, J=1.9 Hz, 1H, H-1), 4.71 (d,J=11.7 Hz, 1H, benzyl-CH₂), 4.63 (d, J=11.7 Hz, 1H, benzyl-CH₂), 4.49(s, 1H, H-5), 4.12-4.10 (m, 1H, H-3), 4.0 (d, J=1.8 Hz, 1H, H-4), 3.97(dd, J=1.9, 3.7 Hz, 1H, H-2), 3.80 (s, 3H, OCH₃), 3.12 (d, J=11.6 Hz,1H, OH), 1.63 (s, 3H, CH₃), 1.38 (s, 3H, CH₃); ¹³C-NMR (125 MHz, CDCl₃)δ 169.4, 137.1, 128.8, 128.5, 128.0, 112.1, 96.5, 75.5, 73.4, 72.9,72.2, 67.4, 52.6, 28.4, 25.7; FAB MS (C₁₇H₂₂O₇) m/z (M)⁺ calcd 338.1365,obsd 338.1357. Further elution (toluene:EtOAc 9:1) afforded 29 (382 mg,20%) as a colorless oil.

Methyl 3-O-benzyl-1,2-O-cyclopentylidene-β-L-idopyranosiduronate 30 andMethyl 3-O-benzyl-1,2-O-cyclopentylidene-β-L-idofuranosiduronate 31

A mixture of (1S)-(+)-camphorsulfonic acid (77 mg, 0.33 mmol) andmethoxycyclopentylidene (3.3 g, 33.5 mmol) in DMF (2 mL) was cooled to0° C. A solution of methyl-3-O-benzyl-L-idopyranosiduronate 23 (1.0 g,3.35 mmol) in DMF (1 mL) was added dropwise under stirring. The mixturewas stirred for 3 h at 0° C. and overnight at room temperature, afterwhich methanol (2 mL) was added and stirred 30 minutes. The reaction wasquenched by adding triethylamine After dilution with EtOAc, the solutionwas washed with water and dried over MgSO₄. After filtration the solventwas removed under reduced pressure and the syrup was purified by flashchromatography on silica gel (toluene:AcOEt 99:1→98:2) to yield 30 (684mg, 1.88 mmol, 56%) as a yellow syrup. [α]²⁴ _(D): +10.9 (c 1.00,CHCl₃); IR (thin film on NaCl) 3534, 2954, 2111, 1764, 1437, 1336, 1120cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.40-7.30 (m, 5H, arom.), 5.35 (d, J=1.9Hz, 1H, H-1), 4.70 (d, J=11.7 Hz, 1H, benzyl-CH₂), 4.63 (d, J=11.7 Hz,1H, benzyl-CH₂), 4.50 (s, 1H, H-5), 4.11-4.09 (m, 1H, H-3), 4.07 (d,J=1.6 Hz, 1H, H-4), 3.88 (dd, J=2.1, 3.6 Hz, 1H, H-2), 3.80 (s, 3H,OCH₃), 3.10 (d, J=11.9 Hz, 1H, OH), 2.14-2.08 (m, 1H, cyclopentenyl),2.23-2.16 (m, 1H, cyclopentenyl), 1.86-1.61 (m, 6H, cyclopentenyl),¹³C-NMR (125 MHz, CDCl₃) δ 169.4, 137.2, 128.8, 128.5, 128.0, 121.5,96.1, 75.7, 73.5, 72.9, 72.1, 67.4, 52.6, 38.3, 36.6, 23.5, 23.4; FAB MS(C₁₉H₂₄O₇) m/z (M)⁺ calcd 364.1522, obsd 364.1530. Further elution(toluene:EtOAc 9:1) afforded methyl3-O-benzyl-1,2-O-cyclopentylidene-α-D-idofuranosiduronate 31 (219 mg,0.6 mmol, 18%) as a colorless oil. [α]²⁴ _(D): −13.9 (c 1.00, CHCl₃); IR(thin film on NaCl) 3485, 2954, 2874, 1738, 1453, 1338, 1207, 1120 cm⁻¹;¹H-NMR (500 MHz, CDCl₃) δ 7.38-7.30 (m, 5H, arom.), 5.97 (d, J=4.1 Hz,1H, H-1), 4.74 (d, J=11.6 Hz, 1H, benzyl-CH₂), 4.64 (dd, J=1.2, 4.1 Hz,1H, H-2), 4.57-450 (m, 2H, H-3, H-5), 4.53 (d, J=11.4 Hz, 1H,benzyl-CH₂,), 4.20 (dd, J=1.5, 4.7 Hz, 1H, H-4), 3.74 (s, 3H, OCH₃),3.30 (d, J=3.3 Hz, 1H, OH), 2.00-1.91 (m, 1H, cyclopentenyl), 1.81-1.65(m, 6H, cyclopentenyl); ¹³C-NMR (100 MHz, CDCl₃) δ 172.3, 137.0, 128.7,128.3, 128.1, 122.2 105.1, 83.5, 83.0, 80.4, 72.5, 70.1, 52.8, 37.3,37.1, 23.4, 23.3; FAB MS (C₁₉H₂₄O₇) m/z (M)⁺ calcd 364.1522, obsd364.1534.

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-isopropylidene-α-D-glucopyranosiduronate 32

Coupling of 11 and 24

Compound 11 (1.87 g, 3.14 mmol) and 24 (850 mg, 2.51 mmol) werecoevaporated with toluene (3×) and dissolved in anhydrousdichloromethane (50 mL). Freshly activated powdered 4 Å molecular sieves(1 g) were added and the mixture was stirred at room temperature for 1h. The reaction mixture was cooled to −78° C. andtert-butyldimethylsilyl trifluoromethanesulfonate (72 μL, 0.314 mmol)was added dropwise. The mixture was warmed to room temperature over 2.5h. Triethylamine (3 mL) was added, the mixture was filtered through apad of Celite and the solvent was removed under reduced pressure. Flashchromatography on silica gel (hexanes:EtOAc 85:15) afforded 32 (1.68 g,2.18 mmol, 86%) as a colorless foam.

Coupling of 10 and 24

Compound 10 (540 mg, 1.19 mmol) and 24 (362 mg, 1.07 mmol) werecoevaporated with toluene (3×), dissolved in Et₂O (20 ml) and cooled to0° C. To this mixture freshly activated 4 Å molecular sieves (1 g),SnCl₂ (229 mg, 1.21 mmol) and AgClO₄ (250 mg, 1.21 mmol) were added.This mixture was stirred for 90 min at 0° C., then warmed to 12° C. over22 h. The mixture was filtered through a pad of Celite, washed with sat.NaHCO₃ and brine. The organic layer was dried over Na₂SO₄ and afterfiltration the solvent was removed under reduced pressure. Flashchromatography on silica gel (hexanes:EtOAc 9:1) afforded 32 (660 mg,0.86 mmol, 80%) as a colorless foam. [α]²⁴ _(D): +97.2 (c 2.55, CH₂Cl₂);IR (thin film on NaCl) 3032, 2953, 2930, 2858, 2106, 1746, 1455, 1372,1250 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.40-7.27 (m, 10H, arom.), 5.78 (d,J=3.7 Hz, 1H, H-1B), 5.17 (d, J=3.7 Hz, 1H, H-1A), 4.88 (d, J=11.0 Hz,1H, benzyl-CH₂), 4.78 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.68 (s, 2H,benzyl-CH₂), 4.59 (d, J=6.1 Hz, 1H, H-5B), 4.38 (dd, J=2.1, 11.9 Hz, 1H,H-6Aa), 4.25-4.21 (m, 2H, H-2B, H-4B), 4.09-4.05 (m, 2H, H-6Ab, H-3B),3.89-3.85 (m, 1H, H-5A), 3.74 (dd, J=8.5, 10.4 Hz, 1H, H-3A), 3.71 (s,3H, OCH₃), 3.63 (dd, J=8.5, 9.8 Hz, 1H, H-4A), 3.25 (dd, J=3.7, 10.4 Hz,1H, H-2A), 2.08 (s, 3H, acetyl-CH₃), 1.63 (s, 3H, isopropylidene-CH₃),1.39 (s, 3H, isopropylidene-CH₃), 0.89 (s, 9H, tert-butyl), 0.02 (s, 3H,CH₃), 0.00 (s, 3H, CH₃); ¹³C-NMR (MHz, CDCl₃) δ 170.9, 170.1, 138.1,137.3, 128.6, 128.4, 128.2, 128.0, 127.7, 127.5, 111.0, 98.2, 95.7,80.0, 76.0, 75.6, 75.1, 73.9, 72.3, 71.9, 71.3, 71.2, 63.6, 62.9, 52.5,27.6, 26.0, 25.9, 21.0, 18.1, −3.6, −4.8; FAB MS (C₃₈H₅₃N₃O₁₂Si) m/z(M)⁺ calcd 771.3399, obsd 771.3386.

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-cyclopentylidene-α-D-glucopyranosiduronate 33

From 11

Compound 11 (870 mg, 1.46 mmol) and 26 (425 mg, 1.17 mmol) werecoevaporated with toluene (3×) and dissolved in CH₂Cl₂ (20 mL). Freshlyactivated powdered 4 Å molecular sieves (500 mg) were added and themixture was stirred at room temperature for 30 min. The mixture wascooled to −25° C. and tert-butyldimethylsilyl trifluoromethanesulfonate(33 μL, 0.146 mmol) was added dropwise. The reaction mixture was warmedto room temperature and stirred overnight. Triethylamine (3 mL) wasadded, the mixture was filtered through a pad of Celite and the solventwas removed under reduced pressure. Flash chromatography on silica gel(hexanes:EtOAc 95:5→9:1) afforded 33 (744 mg, 0.93 mmol, 80%) as acolorless foam. From 10: Compound 10 (610 mg, 1.35 mmol) and 26 (402 mg,1.10 mmol) were coevaporated with toluene (3×), dissolved in Et₂O (20ml) and cooled to 0° C. To this mixture freshly activated powdered 4 Åmolecular sieves (1 g), SnCl₂ (260 mg, 1.37 mmol) and AgClO₄ (290 mg,1.40 mmol) were added. The mixture was stirred for 8 h at 0° C., thenwarmed to 12° C. over 8 h. The mixture was filtered through a pad ofCelite, washed with sat. NaHCO₃ and brine. The organic layer was driedover Na₂SO₄ and after filtration the solvent was removed under reducedpressure. Flash chromatography on silica gel (hexanes:EtOAc 95:5→9:1)afforded 33 (693 mg, 0.87 mmol, 79%) as a colorless foam. [α]²⁴ _(D):+87.5 (c 1.20, CH₂Cl₂); IR (thin film on NaCl) 3025, 2954, 2849, 2105,1746, 1455, 1250 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.40-7.27 (m, 10H,arom.), 5.74 (d, J=4.0 Hz, 1H, H-1B), 5.15 (d, J=3.4 Hz, 1H, H-1A), 4.87(d, J=11.0 Hz, 1H, benzyl-CH₂), 4.79 (d, J=11.0 Hz, 1H, benzyl-CH₂),4.70 (d, J=11.6 Hz, 1H, benzyl-CH₂), 4.67 (d, J=11.9 Hz, 1H,benzyl-CH₂), 4.59 (d, J=6.7 Hz, 1H, H-5B), 4.38 (dd, J=2.1, 11.9 Hz, 1H,H-6Aa), 4.26 (dd, J=3.1, 6.7 Hz, 1H, H-4B), 4.14 (dd, J=3.4, 4.0 Hz, 1H,H-2B), 4.08-4.04 (m, 2H, H-6Ab, H-3B), 3.89-3.84 (m, 1H, H-5A), 3.74(dd, J=8.5, 10.4 Hz, 1H, H-3A), 3.71 (s, 3H, OCH₃), 3.64 (dd, J=9.2 Hz,J=8.9 Hz, 1H, H-4A), 3.24 (dd, J=3.7, 10.4 Hz, 1H, H-2A), 2.08-2.00 (m,5H, acetyl-CH₃, cyclopentenyl), 1.79-1.62 (m, 6H, cyclopentenyl), 0.89(s, 9H, tert-butyl), 0.02 (s, 3H, CH₃), 0.00 (s, 3H, CH₃); ¹³C-NMR (125MHz, CDCl₃) δ 171.0, 170.2, 138.1, 137.3, 128.7, 128.5, 128.2, 128.0,127.8, 127.5, 120.6, 98.0, 95.5, 80.0, 75.7, 75.3, 75.2, 74.0, 72.3,71.4, 71.2, 71.2, 63.6, 62.9, 52.6, 37.0, 36.9, 26.0, 23.8, 23.3, 21.1,18.1, −3.5, −4.8; FAB MS (C₄₀H₅₅N₃O₁₂Si) m/z (M)⁺ calcd 797.3555, obsd797.3578.

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-D-glucopyranosiduronate 34

From 32

Compound 32 (1.68 g, 2.18 mmol) was dissolved in dichloroacetic acid(75% aqueous, 20 mL) and stirred at room temperature for 1 h. Thereaction mixture was added slowly to sat. NaHCO₃ and extracted withCH₂Cl₂ (3×). The organic layer was dried over Na₂SO₄ and afterfiltration the solvent was removed under reduced pressure. Flashchromatography on silica gel (hexanes:EtOAc 1:1) afforded 34 (1.29 g,1.86 mmol, 81%) colorless foam. From 33: Compound 33 (1.19 g, 1.49 mmol)was dissolved in dichloroacetic acid (50% aqueous, 15 mL) and stirred atroom temperature for 2 h. The reaction mixture was added slowly to sat.NaHCO₃ and extracted with CH₂Cl₂ (3×). The organic layer was dried overNa₂SO₄ and after filtration the solvent was removed under reducedpressure. Flash chromatography on silica gel (hexanes:EtOAc 1:1)afforded 34 (980 mg, 1.34 mmol, 90%) as a colorless foam. FAB MS(C₃₅H₄₉N₃O₁₂Si) m/z (M)⁺ calcd 731.3086, obsd 731.3107.

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-isopropylidene-α-D-glucopyranosiduronate 35

Compound 18 (946 mg, 1.65 mmol) and 24 (451 mg, 1.33 mmol) werecoevaporated with toluene (3×) and dissolved in CH₂Cl₂ (30 mL). Freshlyactivated powdered 4 Å molecular sieves (700 mg) were added and themixture was stirred at room temperature for 1 h. The mixture was cooledto −78° C. and tert-butyldimethylsilyl trifluoromethanesulfonate (38 μL,0.165 mmol) was added dropwise. The reacion mixture was warmed to roomtemperature and stirred overnight. Triethylamine (3 mL) was added andthe mixture was filtered through a pad of Celite and the solvent wasremoved under reduced pressure. Flash chromatography (hexanes:EtOAc 8:2)afforded 35 (828 mg, 1.11 mmol, 83%) as a colorless oil. [α]²⁴ _(D):+58.0 (c 1.53, CH₂Cl₂); IR (thin film on NaCl) 3029, 2938, 2017, 1743,1454 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.41-7.27 (m, 15H, arom.), 5.72 (d,J=4.0 Hz, 1H, H-1B), 5.14 (d, J=3.7 Hz, 1H, H-1A), 4.90 (d, J=10.7 Hz,1H, benzyl-CH₂), 4.87 (d, J=10.7 Hz, 1H, benzyl-CH₂), 4.86 (d, J=11.0Hz, 1H, benzyl-CH₂), 4.70 (s, 2H, benzyl-CH₂), 4.59 (d, J=11.0 Hz, 1H,benzyl-CH₂), 4.48 (d, J=7.3 Hz, 1H, H-5B), 4.30 (dd, J=2.4, 11.9 Hz, 1H,H-6Aa), 4.28-4.22 (m, 3H, H-2B, H-4B, H-6Ab), 4.06 (at, J=3.7 Hz, 1H,H-3B), 4.00-3.92 (m, 2H, H-3A, H-5A), 3.73 (s, 3H, OCH₃), 3.54 (dd,J=8.8, 10.1 Hz, 1H, H-4A), 3.30 (dd, J=3.7, 10.4 Hz, 1H, H-2A), 2.05 (s,3H, acetyl-CH₃), 1.62 (s, 3H, isopropylidene-CH₃), 1.39 (s, 3H,isopropylidene-CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 171.4, 170.7, 138.4,138.2, 137.9, 129.3, 129.2, 128.8, 128.7, 128.7, 128.6, 128.5, 111.7,98.3, 96.7, 80.5, 78.5, 77.1, 76.3, 76.1, 75.7, 74.4, 72.8, 72.1, 70.6,63.9, 63.2, 53.2, 28.0, 26.5, 21.5; FAB MS (C₃₉H₄₅N₃O₁₂) m/z (M)⁺ calcd747.3003, obsd 747.3001.

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-cyclopentylidene-α-D-glucopyranosiduronate 36

Compound 18 (855 mg, 1.50 mmol) and 26 (436 mg, 1.20 mmol) werecoevaporated with toluene (3×) and dissolved in CH₂Cl₂ (20 mL). Freshlyactivated powdered 4 Å molecular sieves (250 mg) were added and themixture was stirred at room temperature for 1 h. The mixture was cooledto −20° C. and tert-butyldimethylsilyl trifluoromethanesulfonate (100μL, 0.44 mmol) was added dropwise. The reacion mixture was stirred for2.5 h at −20° C. Triethylamine (3 mL) was added, the mixture wasfiltered through a pad of Celite and the solvent was remove dunderreduced pressure. Flash chromatography on silica gel (hexanes:EtOAc85:15→8:2) afforded 36 (760 mg, 0.98 mmol, 82%) as a colorless oil.[α]²⁴ _(D): +62.3 (c 1.07, CH₂Cl₂); IR (thin film on NaCl) 2948, 2107,1743, 1454, 1362 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.40-7.27 (m, 15H,arom), 5.69 (d, J=4.0 Hz, 1H, H-1B), 5.12 (d, J=3.7 Hz, 1H, H-1A), 4.90(d, J=10.7 Hz, 1H, benzyl-CH₂), 4.87 (d, J=10.7 Hz, 1H, benzyl-CH₂),4.86 (d, J=11.3 Hz, 1H, benzyl-CH₂), 4.70 (s, 2H, benzyl-CH₂), 4.60 (d,J=11.0 Hz, 1H, benzyl-CH₂), 4.48 (d, J=7.3 Hz, 1H, H-5B), 4.31 (dd,J=2.1, 12.2 Hz, 1H, H-6Aa), 4.28-4.24 (m, 2H, H-4B, H-6Ab), 4.18 (dd,J=3.7, 4.0 Hz, 1H, H-2B), 4.06 (at, J=3.4 Hz, 1H, H-3B), 4.01-3.93 (m,2H, H-3A, H-5A), 3.74 (s, 3H, OCH₃), 3.55 (dd, J=8.8, 10.0 Hz, 1H,H-4A), 3.29 (dd, J=3.7, 10.4 Hz, 1H, H-2A), 2.09-2.03 (m, 5H,acetyl-CH₃, cyclopentenyl), 1.79-1.68 (m, 6H, cyclopentenyl); ¹³C-NMR(125 MHz, CDCl₃) δ 171.4, 170.7, 138.4, 138.2, 137.9, 129.3, 129.2,128.8, 128.7, 128.7, 128.6, 128.5, 121.2, 98.1, 96.4, 80.5, 78.5, 76.8,76.1, 76.0, 75.7, 74.5, 72.8, 71.6, 70.6, 63.8, 63.2, 53.2, 37.4, 37.3,24.4, 23.8, 21.5; FAB MS (C₄₁H₄₇N₃O₁₂) m/z (M)⁺ calcd 773.3160, obsd773.3179.

6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl-3-O-benzyl-D-glucopyranosiduronate37

From 35

Compound 35 (315 mg, 0.42 mmol) was dissolved in dichloroacetic acid(75% aqueous, 4 mL) and stirred at room temperature for 2 h. Thereaction mixture was added slowly to sat. NaHCO₃ and extracted withCH₂Cl₂ (3×). The organic layer was dried over Na₂SO₄ and afterfiltration the solvent was remove under reduced pressure. Flashchromatography on silica gel (hexanes:EtOAc 1:1) afforded 37 (250 mg,0.35 mmol, 84%) as a colorless foam. From 36: Compound 36 (836 mg, 1.08mmol) was dissolved in dichloroacetic acid (75% aqueous 12 mL) andstirred at room temperature for 2 h. The reaction mixture was addedslowly to sat. NaHCO₃ and extracted with CH₂Cl₂ (3×). The organic layerwas dried over Na₂SO₄ and after filtration the solvent was removed underreduced pressure. Flash chromatography (hexanes:EtOAc 1:1) afforded 37(619 mg, 0.87 mmol, 81%) as a colorless foam. FAB MS (C₃₆H₄₁N₃O₁₂) m/Z(M)⁺ calcd 707.2690, obsd 707.2678.

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-isopropylidene-α-L-idopyranosiduronate 39

A mixture of 11 (1.38 g, 2.32 mmol) and 28 (627 mg, 1.9 mmol) wascoevaporated with toluene (3×) and dried under vacuum for 1 h. Themixture was dissolved in CH₂Cl₂ (20 mL) and was stirred for 30 min atroom temperature under argon in the presence of freshly activatedpowdered 4 Å molecular sieves (800 mg). After cooling the mixture to−30° C., tert-butyldimethylsilyltrifluoromethanesulfonate (1 M in dryCH₂Cl₂, 230 μL, 0.23 mmol) was added dropwise. The mixture was warmed toroom temperature over 1 h. Triethylamine (4 mL) was added, the mixturewas filtered through a pad of Celite and the solvent was removed underreduced pressure. The residue was purified by silica gel columnchromatography (toluene:EtOAc 95:5→92:8) to yield 39 (1.30 g, 1.68 mmol,91%) as a colorless glass. [α]²⁴ _(D): +93.3 (1, CHCl₃); IR (thin filmon NaCl) 2890, 2100, 1740, 1020 cm-1; 1H-NMR (500 MHz, CDCl3) 7.38-1.20(m, 10H, arom.), 5.28(d, J=2.4 Hz, 1H, H-1B), 4.93 (d, J=3.0 Hz, 1H,H-1A), 4.76 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.65 (d, J=11.0 Hz, 1H,benzyl-CH₂), 4.61 (s, 2H, H-6Aa, benzyl-CH₂) 4.41-4.36 (m, 2H, H-3A,benzyl-CH₂), 4.13 (t, J=2.1 Hz, 1H, H-3B), 3.99 (s, 1H, H-5B), 3.95 (dd,J=3.0, 12.2 Hz, 1H, H-6Ab), 3.91 (s, 1H, H-4B, H-6Ab), 3.74-3.71 ( m,1H, H-4A), 3.71 (s, 3H, OCH₃), 3.61-3.59 (m, 2H, H-2B, H-5A) 3.30 (dd,J=3.3, 9.5 Hz, 1H, H-2A), 2.08 (s, 3H, acetyl-CH₃), 1.98 (s, 3H,acetyl-CH₃), 1.57 (s, 3H, isopropyl-CH₃), 1.33 (s, 3H, isopropyl-CH₃),0.78 (s, 9H, tert-butyl), −0.10 (s, 6H, CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ170.8, 169.0, 138.2, 137.1 128.8, 128.4, 128.0 127.9, 127.7, 127.6,112.2, 98.1, 97.1, 80.1, 97.3, 75.4, 75.2, 73.5, 72.9, 72.8, 71.5, 71.3,70.6, 68.9, 64.6, 62.5, 52.5, 28.3, 26.3, 26.0, 21.1, 18.1, −3.6, −5.0;FAB MS (C₃₈H₅₃N₃O₁₂Si) m/z (M)⁺ calcd 771.3399, obsd 771.3415.

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-isopropylidene-β-L-idopyranosiduronate 40

A mixture of 38 (500 mg, 0.91 mmol) and 28 (241 mg, 0.71 mmol) wascoevaporated with toluene (3×) and dried under vacuum for 1 h. Themixture was dissolved in CH₂Cl₂ (15 mL) and was stirred for 30 min atroom temperature under argon in the presence of freshly activatedpowdered 4 Å molecular sieves (400 mg). After cooling the mixture to−30° C., tert-butyldimethylsilyltrifluoromethanesulfonate (0.1 M in dryCH₂Cl₂, 1 mL, 0.1 mmol) was added dropwise. The mixture was warmed to 0°C. over 1 h. Triethylamine (1.8 mL) was added, the mixture was filteredthrough a pad of Celite and the solvent was removed under reducedpressure. The residue was purified by silica gel column chromatography(toluene:EtOAc 95:5→92:8) to yield 40 (463 mg, 0.64 mmol, 90%) as acolorless glass. [α]²⁴ _(D): +87.4 (c 100, CHCl₃); IR (thin film onNaCl) 2880, 2086, 1731, 1440, 1054 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ7.38-7.32 (m, 5H, arom.), 5.34 (d, J=2.1 Hz, 1H, H-1B), 5.32-5.36 (dd,1H, H-3A), 4.93 (d, J=3.0 Hz, 1H, H-1A), 4.70 (d, J=11.6 Hz, 1H,benzyl-CH₂), 4.67 (d, J=11.6 Hz, 1H, benzyl-CH₂), 4.50 (dd, J=2.1, 12.5Hz, 1H, H-6Aa), 4.42 (d, J=1.5 Hz, 1H, H-5B), 4.27 (t, J=2.1 Hz, 1H,H-3B), 4.06-4.02 (m, 2H, H-4B, H-6Ab), 3.98-3.94 (m, 2H, H-2B, H-5A),3.80-3.74 (m, 1H, H-4A), 3.79 (s, 3H, OCH₃), 3.17 (dd, J=10.7, 3.4 Hz,1H, H-2A), 2.08 (s, 3H, acetyl-CH₃), 2.00 (s, 3H, acetyl-CH₃), 1.64 (s,3H, isopropyl-CH₃), 1.39 (s, 3H, isopropyl-CH₃), 0.81 (s, 9H,tert-butyl), 0.03 (s, 3H, CH₃), 0.01 (s, 3H, CH₃); ¹³C-NMR (125 MHz,CDCl₃) δ 170.6, 169.8, 169.1, 137.3, 128.8, 128.4, 128.1, 112.5, 99.1,97.3, 75.7, 74.8, 73.9, 73.0, 72.8, 71.3, 71.3, 68.9, 62.4, 62.3, 52.7,28.1, 26.2, 25.7, 21.5, 21.1, 18.0, −3.9, −4.9; FAB MS (C₃₃H₄₉N₃O₁₃Si)m/z (M)⁺ calcd 723.3035, obsd 723.3058.

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-cyclopentylidene-β-L-idopyranosiduronate 41

A mixture of 38 (200 mg, 0.36 mmol) and 13 (95 mg, 0.28 mmol) wascoevaporated with toluene (3×) and dried under vacuum for 1 h. Themixture was dissolved in CH₂Cl₂ (5 mL) and was stirred for 30 min atroom temperature under argon in the presence of freshly activatedpowdered 4 Å molecular sieves (150 mg). After cooling the mixture to−30° C., tert-butyldimethylsilyltrifluoromethanesulfonate (0.1 M in dryCH₂Cl₂, 0.4 mL, 0.04 mmol) was added dropwise. The mixture was warmed to0° C. over 1 h. Triethylamine (0.7 mL) was added, the mixture wasfiltered through a pad of Celite and the solvent was removed underreduced pressure. The residue was purified by silica gel columnchromatography (toluene:EtOAc 96:3→90:10) to yield 41 (217 mg, 0.29mmol, 88%) as a colorless glass. [α]²⁴ _(D): +75.6 (c 1.00, CH₂Cl₂); IR(thin film on NaCl) 2955, 2858, 2109, 1744, 1455 cm⁻¹; ¹H-NMR (400 MHz,CDCl₃) δ 7.38-7.30 (m, 5H, arom.), 5.34-5.29 (m, 2H, H-1B, H-3A), 4.68(t, J=12.0 Hz, 2H, benzyl-CH₂), 4.51 (m, 1H, H-6Aa), 4.46 (d, J=1.5 Hz1H, H-5B), 4.28 (dd, J=2.1, 2.9 Hz, 1H, H-3H), 4.13 (t, 1H, H-4B),4.07-4.02 (m, 2H, H-5A, H-6Ab), 3.87 (t, 1H, H-2B), 3.80-3.74 (m, 1H,H-4A), 3.77 (s, 3H, OCH₃), 3.07 (dd, J=3.4, 10.6 Hz, 1H, H-2A), 2.13 (s,3H, acetyl-CH₃), 2.06 (s, 3H, acetyl-CH₃), 2.09-04 (m, 2H,cyclopentenyl), 1.78-1.63 (m, 6H, cyclopentenyl), 0.82 (s, 9H,tert-butyl), 0.04 (s, 3H, CH₃), 0.02 (s, 3H, CH₃); ¹³C-NMR (125 MHz,CDCl₃) δ 170.6, 169.8, 169.1, 137.3, 128.7, 128.3, 121.7, 98.9, 96.9,76.9, 75.8, 74.4, 74.1, 73.0, 72.7, 71.1, 71.0, 68.9, 62.3, 62.1, 52.6,37.6, 36.9, 25.7, 23.5, 21.5, 21.0, 18.0, −3.9, −4.9; FAB MS(C₃₅H₅₁N₃O₁₃Si) m/z (M)⁺ calcd 749.3191, obsd 749.3197.

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-L-idopyranosiduronate 42

A solution of 39 (1.20 g, 1.55 mmol) in dichloroacetic acid (40 mL, 60%aq) was stirred at room temperature for 3 h, diluted with water andneutralized with NaHCO₃ (24 g). The aqueous phase was extracted threetimes with CH₂Cl₂. The combined organic phases were dried over MgSO₄.After filtration the solvent was removed under reduced pressure toafford 42 (1.05 g, 1.4 mmol, 92%) as an essentially pure white solid.Compound 42 can be further purified by silica gel column chromatography.(hexane:EtOAc 70:30). FAB MS (C₃₅H₄₉N₃O₁₂Si) m/z (M)⁺ calcd 731.3086,obsd 731.3002.

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-L-idopyranosiduronate 43

From 40

A solution of 40 (770 mg, 1.06 mmol) in dichloroacetic acid (10 mL, 60%aq) was stirred at room temperature for 3 h, diluted with water andneutralized with NaHCO₃ (7 g). The aqueous phase was extracted threetimes with CH₂Cl₂ and the combined organic phases were dried over MgSO₄.After filtration the solvent was removed under reduced pressure toafford 43 (647 mg, 0.95 mmol, 89%) as a white solid. Compound 43 can befurther purified by silica gel column chromatography (hexane:EtOAc70:30). FAB MS (C₃₀H₄₅N₃O₁₃Si) m/z (M)⁺ calcd 683.2722, obsd 683.2743.From 41: A solution of 41 (200 mg, 0.27 mmol) in dichloroacetic acid (3mL, 60% aq) was stirred at room temperature for 3 h, diluted with waterand neutralized with NaHCO₃ (7 g). The aqueous phase was washed threetimes with CH₂Cl₂. The combined organic phases were dried over MgSO₄.After filtration the solvent was removed under reduced pressure toafford 43 (160 mg, 0.23 mmol, 88%) as an essentially pure white solid.

6-O-Acetyl-2-azido3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-monochloroacetyl-α/β-glucopyranosiduronate 44

Pyridine (2.1 mL, 24 mmol), monochloroacetic anhydride (3.0 g, 9.00mmol) and DMAP (19 mg, 0.157 mmol) were added to a solution of 34 (1.3g, 1.57 mmol) in CH₂Cl₂ (21 mL). The solution was stirred at roomtemperature for 6 h, water was added and the mixture was stirred for oneadditional hour. The organic phase was washed with sat. NaHCO₃, waterand aqueous HCl (10%), dried over MgSO₄ and filtered. The solvent wasremoved under reduced pressure and the residue was purified by silicagel column chromatography (hexane:EtOAc 90:20) to yield 44 (1.5 g, 1.7mmol, 96%) as a colorless syrup. FAB MS (C₃₉H₅₁Cl₂N₃O₁₄Si) m/z (M)⁺calcd 883.2517, obsd 883.2506.

6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-1,2-O-monochloroacetyl-α/β-D-glucopyranosiduronate 45

Pyridine (0.65 mL, 7.4 mmol), monochloroacetic anhydride (820 mg, 2.5mmol) and DMAP (7 mg, 0.06 mmol) were added to a solution of 37 (400 mg,0.6 mmol) in CH₂Cl₂ (6.5 mL). The solution was stirred at roomtemperature for 6 h, water was added and the mixture was stirred for oneadditional hour. The organic phase was washed with saturated solution ofNaHCO₃, water and 10% HCl, dried over MgSO₄ and filtered. The solventwas removed under reduced pressure and the residue was purified bysilica gel column chromatography (hexane:EtOAc 90:20) to yield 45 (452mg, 0.53 mmol 93%) as a colorless syrup. FAB MS (C₄₀H₄₃Cl₂N₃O₁₄) m/z(M)⁺ calcd 859.2122, obsd 859.2113.

6-O-Acetyl-2-azido3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl-3-O-benzyl-2-O-monochloroacetyl-α-glucopyranosyluronatetrichloroacetimidate 46

From 44

Benzylamine (70 μL, 0.63 mmol) was added in three portions, every 2 h,to a solution of 44 (1 g, 1.2 mmol) Et₂O (40 mL) at 0° C. and keptovernight at −20° C. The mixture was diluted with CH₂Cl₂, filtered andwashed with aqueous HCl (10%). The organic phase was dried over MgSO₄,filtered and the solvent was removed under reduced pressure. Flashchromatography on silica gel (hexane:EtOAc 90:10→80:20) afforded6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-D-glucopyranosiduronate (704 mg, 0.87mmol, 75%) as a white solid. FAB MS (C₃₇H₅₀ClN₃O₁₃Si) m/z (M)⁺ calcd807.2801, obsd 807.2796.

From 50

A mixture of tetrabutylammonium fluoride (1.0 M in THF, 0.6 mL) andglacial acetic acid (55 μl, 0.9 mmol) was added dropwise to a solutionof 50 (539 mg, 0.58 mmol) in THF (6 mL) under nitrogen at 0° C. Thereaction mixture was warmed to room temperature, stirred for 1.5 h andquenched with brine. After dilution with CH₂Cl₂, the two phases wereseparated. The organic phase was dried over MgSO₄, filtered and thesolvent was removed under reduced pressure. The crude material waspurified by silica gel column chromatography (hexane:EtOAc 90:10→80:20)to yield6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-α/β-D-glucopyranosiduronate (198 mg,43%) as a white solid.

A solution of6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-D-glucopyranosiduronate (540 mg, 0.7mmol) and trichloroacetonitrile (2 mL, 19.2 mmol) in CH₂Cl₂ (14 mL)containing freshly activated 4 Å molecular sieves (100 mg), was stirred30 minutes at room temperature. After cooling to 0° C., before DBU (45μl, 0.3 mmol) was added. The mixture was allowed to reach roomtemperature after 1 h the mixture was filtered through a pad of Celiteand concentrated. The residue was purified by silica gel columnchromatography (hexane:EtOAc 85:15) to yield 46 (546 mg, 0.57 mmol, 85%)as a white solid. [α]²⁴ _(D): +81.5 (c 1.00, CHCl₃); IR (thin film onNaCl) 2930, 2106, 1745, 1678, 1252, 1029 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ8.69 (s, 1H, NH), 7.36-7.26 (m, 10H, H-arom.), 6.56 (d, J=3.2 Hz, H-1B),5.60 (d, J=3.6 Hz, 1H, H-1A), 5.14-5.19 (m, 1H, H-2B), 4.96-4.78 (m, 4H,benzyl-CH₂), 4.49 (d, 1H, H-5B), 4.35 (dd, J=11.8 Hz, 1H, H-6aA),4.28-4.25 (m, 2H, H-4B, H-3B), 4.04 (dd, J=3.8, 12.1 Hz, 1H, H-6bA),3.99 (d, J=9.5 Hz, H-5B), 3.84-3.76 (m, 2H, CH₂Cl), 3.79 (s, 3H, OCH₃),3.72-3.61 (m, 2H, H-3A, H-5A), 3.50 (m, 1H, H-4A), 3.26 (dd, J=3.6, 10.2Hz, 1H, H-2A), 2.10 (s, 3H, COCH₃), 0.88 (s, 9H, C(CH₃)₃)₃), −0.01 (s,6H, SiCH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 171.0, 168.5, 166.5, 160.8,138.0, 137.9, 128.8, 128.5, 128.1, 127.8, 127.5, 127.4, 98.2, 92.9,80.1, 79.6, 75.5, 75.3, 75.0, 74.3, 72.6, 71.3, 70.9, 63.7, 62.5, 53.1,40.3, 26.0, 21.1, 18.2, −3.5, −4.8; FAB MS (C₃₉H₅₀Cl₄N₄O₁₃Si) m/z (M)⁺calcd 950.1898, obsd 950.1892.

O-(6-O-Acetyl-2-azido3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl-3-O-benzyl-2-O-monochloroacetyl-α-D-glucopyranosyduronate)trichloroacetimidate 47

Benzylamine (70 μL, 0.63 mmol) was added in three portions, every 2 h,to a solution of 45 (400 mg, 0.46 mmol) in Et₂O (25 mL) at 0° C. andkept overnight at −20° C. The mixture was diluted with CH₂Cl₂, filteredand washed with aqueous HCl (10%). The organic phase was dried overMgSO₄, filtered and the solvent was removed under reduced pressure.Flash chromatography on silica gel (hexane:EtOAc 90:10→80:20) afforded6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-D-glucopyranosiduronate (277 mg, 0.35mmol, 76%) as a white solid. FAB MS (C₃₈H₄₂ClN₃O₁₃Si) m/z (M)⁺ calcd783.2406 obsd 783.2400.

A solution of6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-D-glucopyranosiduronate (226 mg, 0.292mmol) and trichloroacetonitrile (0.790 mL, 7.6 mmol) in CH₂Cl₂ (7 mL)containing freshly activated 4 Å molecular sieves (100 mg), was stirred30 minutes at room temperature. After cooling to 0° C., DBU (5 μl, 0.03mmol) was added. The mixture was allowed to reach room temperature andafter 1 h it was filtered through a pad of Celite and concentrated. Theresidue was purified by silica gel column chromatography (hexane:EtOAc85:15) to yield 47 (240 mg, 0.26 mmol, 90%) as a white solid. FAB MS(C₄₀H₄₂Cl₄N₄O₁₃Si) m/z (M)⁺ calcd 926.1502, obsd 926.1514; ¹H-NMR (500MHz, CDCl₃) 8.70 (s, 1H, NH), 7.38-7.27 (m, 15H, arom.), 6.56 (d, J=3.4Hz, 1H, H-1B) 5.53 (d, J=3.7 Hz, 1H, H-1A), 5.17-5.14 (m, 1H, H-2B),4.99-4.78 (m, 5H, benzyl-CH₂), 4.46-4.42 (m, 2H, H-4B, benzyl-CH₂),4.28-4.19 (m, 4H, H-3B, H-5B, H-6Aa, H-6Ab), 3.97-3.90 (m, 1H, H-3A),3.84-3.70 (m, 2H, CH₂Cl), 3.77 (s, 3H, OCH₃), 3.62-3.49 (m, 2H, H-4A,H-5A), 3.32 (dd, J=3.8, 10.4 Hz, 1H, H-2A), 2.04 (s, 3H, acetyl-CH₃);¹³C-NMR (100 MHz, CDCl₃) 170.8, 168.4, 166.5, 160.7, 137.9, 137.8,137.7, 137.6, 129.0, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0,127.5, 98.4, 92.9, 90.7, 80.1, 79.3, 77.6, 77.5, 75.7, 75.6, 75.2, 75.1,73.5, 72.6, 70.1, 63.3, 62.3, 53.2, 40.3, 21.0.

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate 48

Compound 34 (1.12 g, 1.53 mmol) and imidazole (208 mg, 3.05 mmol) weredissolved in CH₂Cl₂ (10 mL) and cooled to −15° C.tert-Butyldimethylsilyl-chloride (253 mg, 1.68 mmol) was added to themixture and stirring was continued at −15° C. After 5 htert-butyldimethylsilylchloride (125 mg, 0.83 mmol) was added and after6 h imidazole (100 mg, 1.47 mmol) and tert-butyldimethylsilylchloride(253 mg, 1.68 mmol) were added. After 18 h one additional portion oftert-butyldimethylsilylchloride (70 mg, 0.46 mmol) was added. After 40h, water was added and the mixture was warmed to room temperature. Afterdilution with EtOAc the mixture was washed with sat. NaHCO₃ and brine.The organic layer was dried over Na₂SO₄ and after filtration the solventwas removed under reduced pressure. Flash chromatography on silica gel(hexanes:EtOAc 9:1→8:2) afforded tert-butyldimethylsilyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate, (1.08 g, 1.28 mmol, 84%) as acolorless foam. [α]²⁴ _(D): +65.9 (c 1.55, CH₂Cl₂); IR (thin film onNaCl) 3475, 3031, 2953, 2857, 2106, 1747, 1472, 1254 cm⁻¹; ¹H-NMR (500MHz, CDCl₃) δ 7.41-7.27 (m, 10H, arom.), 5.64 (d, J=3.7 Hz, 1H, H-1A),5.07 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.89 (d, J=11.0 Hz, 1H,benzyl-CH₂), 4.83 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.81 (d, J=1.0 Hz, 1H,benzyl-CH₂), 4.59 (d, J=7.3 Hz, 1H, H-1B), 4.35 (dd, J=2.1, 11.9 Hz, 1H,H-6Aa), 4.14 (dd, J=8.8, 9.5 Hz, 1H, H-4B), 4.07 (dd, J=3.7, 12.2 Hz,1H, H-6Ab), 3.99 (d, J=9.8 Hz, 1H, H-5B), 3.79 (s, 3H, OCH₃), 3.76 (at,J=8.8 Hz, 1H, H-3B), 3.69-3.63 (m, 2H, H-3A, H-4A), 3.57 (ddd, J=9.5,9.2, 2.1 Hz, 1H, H-2B), 3.52-3.49 (m, 1H, H-5A), 3.28-3.22 (m, 1H,H-2A), 2.31 (d, J=2.1 Hz, 1H, OH), 2.10 (s, 3H, acetyl-CH₃), 0.92 (s,9H, tert-butyl), 0.89 (s, 9H, tert-butyl), 0.15 (s, 6H, 2×CH₃), 0.00 (s,6H, 2×CH ₃); ¹³C-NMR (125 MHz, CDCl₃) δ 171.0, 168.8, 138.5, 138.1,128.6, 128.5, 127.9, 127.8, 127.7, 127.5, 98.0, 97.7, 83.9, 80.2, 76.6,75.2, 74.9, 74.8, 74.7, 71.0, 70.8, 63.8, 62.6, 52.8, 26.0, 25.9, 21.1,18.2, 18.1, −3.5, −4.1, −4.9, −5.0; FAB MS (C₄₁H₆₃N₃O₁₂Si₂) m/z (M)⁺calcd 845.3950, obsd 845.3925.

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate (998 mg, 1.18 mmol) and DMAP (432mg, 3.54 mmol) were dissolved in CH₂Cl₂ (10 mL) and a solution oflevulinic anhydride (500 mg, 2.34 mmol) in CH₂Cl₂ (5 mL) was added. Thereaction mixture was stirred for 2 h at room temperature andconcentrated. Flash chromatography on silica gel (hexanes:EtOAc 8:2)afforded 48 (1.08 g, 1.14 mmol, 97%) as a colorless foam. ¹H-NMR (400MHz, CDCl₃) δ 7.39-7.24 (m, 10H, arom.), 5.53 (d, J=3.6 Hz, 1H, H-1A),5.04 (dd, J=7.3, 8.8 Hz, 1H, H-2B), 4.90-4.73 (m, 5H, 4×benzyl-CH₂,H-1B), 4.37 (dd J=1.8, 11.9 Hz, 1H, H-6Aa), 4.26 (at, J=9.2, 9.0 Hz, 1H,H-4B), 4.07-4.00 (m, 2H, H-6Ab, H-5B), 3.86 (dd, J=8.8, 8.7 Hz, 1H,H-3B), 3.79 (s, 3H, OCH₃), 3.68-3.61 (m, 2H, H-3A, H-4A), 3.55-3.52 (m,1H, H-5A), 3.25 (dd, J=3.7, 9.9 Hz, 1H, H-2A), 2.68-2.63 (m, 2H,Lev-CH₂), 2.53-2.48 (m, 2H, Lev-CH₂), 2.12 (s, 3H, Lev-CH₃), 2.08 (s,3H, acetyl), 0.89 (s, 9H, tert-butyl), 0.87 (s, 9H, tert-butyl), 0.12(s, 3H, CH₃), 0.10 (s, 3H, CH₃), 0.00 (s, 3H, CH₃), −0.03 (s, 3H, CH₃);¹³C-NMR (100 MHz, CDCl₃) δ 206.0, 171.2, 170.8, 168.5, 138.0, 137.9,128.5, 128.4, 127.8, 127.7, 127.6, 127.4, 97.5, 96.1, 82.5, 80.2, 75.2,75.1, 74.7, 74.5, 74.1, 71.1, 70.8, 63.8, 62.5, 52.7, 37.8, 29.9, 28.0,26.0, 25.6, 21.0, 18.1, 17.9, −3.6, −4.2, −5.0, −5.2; FAB MS(C₄₆H₆₉N₃O₁₄Si₂) m/z (M)⁺ calcd 943.4318, obsd 943.4332.

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl2-O-allyloxycarbonyl-3-O-benzyl-β-D-glucopyranosiduronate 49

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate, (for the experimental procedure,please see the procedure to prepare compound 48) (350 mg, 0.414 mmol)and DMAP (1.01 g, 8.28 mmol) were dissolved in CH₂Cl₂ (10 mL) and cooledto −70° C. Allyloxycarbonylchloride (800 μl, 7.54 mmol) was added inthree equal portions every 2 h. After the addition was complete themixture was warmed to room temperature and stirred overnight. Themixture was poured into EtOAc and washed with 1 N HCl, brine and sat.NaHCO₃. The organic layer was dried over Na₂SO₄, filtered and thesolvent was removed under reduced pressure. Flash chromatography onsilica gel (hexanes:EtOAc 9:1→85:15) afforded 49 (347 mg, 0.373 mmol,90%) as a colorless oil. [α]²⁴ _(D): +82.6 (c 1.04, CH₂Cl₂); IR (thinfilm on NaCl 3037, 2929, 2857, 2106, 1758, 1454, 1252 cm⁻¹; ¹H-NMR (500MHz, CDCl₃) δ 7.39-7.26 (m, 10H, arom.), 5.91-5.82 (m, 1H, alloc), 5.55(d, J=3.7 Hz, 1H, H-1A), 5.35-5.30 (m, 1H, alloc), 5.25-5.22 (m, 1H,alloc), 4.89-4.71 (m, 6H, H-1B, H-2B, 4×benzyl-CH₂), 4.62-4.53 (m, 2H,alloc), 4.34 (dd, J=2.1, 11.9 Hz, 1H, H-6Aa), 4.22 (dd, J=9.5, 9.2 Hz,1H, H-4B), 4.05 (dd, J=3.7, 12.2 Hz, 1H, H-6Ab), 3.99 (d, J=9.8 Hz, 1H,H-5B), 3.85 (d, J=9.2 Hz, 1H, H-3B), 3.80 (s, 3H, OCH₃), 3.68-3.61 (m,2H, H-3A, H-4A), 3.52-3.49 (m, 1H, H-5A), 3.29-3.25 (m, 1H, H-2A), 2.10(s, 3H, acetyl-CH₃), 0.89 (s, 9H, tert-butyl), 0.87 (s, 9H, tert-butyl),0.12 (s, 3H), CH₃), 0.10 (s, 3H, CH₃), 0.00 (s, 6H, 2×CH₃); ¹³C-NMR (125MHz, CDCl₃) δ 171.0, 168.5, 154.1, 138.0, 137.8, 131.4, 128.6, 128.5,127.9, 127.8, 127.7, 127.5, 119.6, 97.8, 96.1, 82.6, 80.2, 79.1, 75.3,75.0, 74.8, 74.6, 71.1, 70.8, 69.0, 63.9, 62.5, 52.9, 26.0, 25.6, 21.1,18.1, 18.0, −3.5, −4.1, −4.9, −5.3; FAB MS (C₄₅H₆₇N₃O₁₄Si₂) m/z (M)⁺calcd 929.4162, obsd 929.4122.

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-β-D-glucopyranosiduronate 50

Pyridine (0.5 mL, 6 mmol), monochloroacetic anhydride (360 mg, 1.00mmol) and DMAP (9 mg, 0.05 mmol) were added to a solution oftert-butyldimethylsilyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(→4)-methyl-3-O-benzyl-β-D-glucopyranosiduronate(for the synthesis see the procedure to prepare compound 48) (439 mg,0.52 mmol) in CH₂Cl₂ (5 mL). The solution was stirred at roomtemperature for 3 h, water was added and the mixture was stirred for oneadditional hour. The organic phase was washed with sat. NaHCO₃, waterand aqueous HCl (10%), dried over MgSO₄ and filtered. The solvent wasremoved under reduced pressure and the residue was purified by silicagel column chromatography (hexanes:EtOAc 90:20) to yield 50 (470 mg,0.51 mmol, 98%) as a colorless syrup. [α]²⁴ _(D): +30.1 (c 1.00, CHCl₃);IR (thin film on NaCl) 2932, 2107, 1745, 1254, 1029, 839 cm⁻¹; ¹H-NMR(500 MHz, CDCl₃) δ 7.37-7.28 (m, 10H, H arom.), 5.50 (d, J=3.4 Hz, 1H,H-1A), 5.05 (dd, J=7.9, 9.1 Hz, H-2B), 4.74 (d, J=7.3 Hz, H-1B), 4.86(d, J=11.0 Hz, 2H, benzyl-CH₂), 4.79 (d, J=11.3 Hz, 1H, benzyl-CH₂),4.68 (d, J=11.3 Hz, 1H, benzyl-CH₂), 4.35 (dd, J=1.5, 11.9 Hz, 1H,H-6aA), 4.29-4.23 (m, 1H, H-4B), 4.04 (dd, J=3.7, 12.2 Hz, 1H, H-6bA),3.99 (d, J=9.5 Hz, H-5B), 3.89-3.83 (m, 2H, H-3B, CH₂Cl), 3.79 (s, 3H,OCH₃), 3.78 (d, J=14.9 Hz, 1H, CH₂Cl), 3.60-3.67 (m, 2H, H-3A, H-5A),3.53-3.51 (m, 1H, H4A), 3.28 (dd, J=3.0, 9.2 Hz, 1H, H-2A), 2.10 (s, 3H,COCH₃), 0.86 (s, 9H, C(CH₃)₃), 0.85 (s, 9H, C(CH₃)₃), 0.10 (s, 3H,SiCH₃), 0.82 (s, 3H, SiCH₃), −0.01 (s, 6H, SiCH₃); ¹³C-NMR (125 MHz,CDCl₃) δ 171.0, 168.4, 165.8, 138.0, 137.8, 128.7, 128.5, 128.0, 127.8,127.7, 127.5, 97.8, 96.0, 82.6, 80.2, 76.4, 75.3, 75.2, 74.7, 74.6,71.3, 70.8, 63.9, 62.5, 52.9, 26.1, 25.6, 21.1, 18.2, 17.9, −3.5, −4.1,−4.9, −5.1; FAB MS (C₄₃H₆₄ClN₃O₁₃Si) m/z (M)⁺ calcd 921.3666, obsd921.366.

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl-3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate 51

Compound 37 (813 mg, 1.15 mmol) and imidazole (310 mg, 4.55 mmol) weredissolved in CH₂Cl₂ (10 mL) and cooled to −15° C. To this mixturetert-butyldimethylsilyl-chloride (241 mg, 1.60 mmol) was added andstirring was continued at −15° C. After 5 h,tert-butyldimethylsilylchloride (50 mg, 0.33 mmol) was added and after16 h another portion of tert-butyldimethylsilylchloride (100 mg, 0.66mmol). After 40 h, water was added and the mixture was warmed to roomtemperature. After dilution with EtOAc the mixture was washed with sat.NaHCO₃ and brine. The organic layer was dried over Na₂SO₄ and afterfiltration the solvent was removed under reduced pressure. Flashchromatography on silica gel (hexanes:EtOAc 9:1→8:2) affordedtert-butyldimethylsilyl(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate (752 mg, 0.92 mmol, 80%) as acolorless foam. [α]²⁴ _(D): +26.2 (c 1.02, CH₂Cl₂); IR (thin film onNaCl) 3376, 3049, 2919, 2861, 2107, 1744, 1454, 1362, 1252 cm⁻¹; ¹H-NMR(500 MHz, CDCl₃) δ 7.42-7.26 (m, 15H, arom. H), 5.60 (d, J=4.0 Hz, 1H,H-1A), 5.08 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.92-4.89 (m, 2H,benzyl-CH₂), 4.86 (d, J=10.7 Hz, 1H, benzyl-CH₂), 4.84 (d, J=11.0 Hz,1H, benzyl-CH₂), 4.58 (d, J=7.3 Hz, 1H, H-1B), 4.57 (d, J=10.7 Hz, 1H,benzyl-CH₂), 4.27-4.25 (m, 2H, H-6Aa, H-6Ab), 4.14 (dd, J=8.9, 9.5 Hz,1H, H-4B), 3.96 (d, J=9.8 Hz, 1H, H-5B), 3.91 (dd, J=8.9, 10.4 Hz, 1H,H-3A), 3.77 (s, 3H, OCH₃), 3.73 (dd, J=9.2, 8.9 Hz, 1H, H-3B), 3.65-3.61(m, 1H, H-5A), 3.59-3.52 (m, 2H, H-2B, H-4A), 3.30 (dd, J=4.0, 10.4 Hz,1H, H-2A), 2.33 (d, J=2.4 Hz, 1H, OH), 2.04 (s, 3H, acetyl-CH₃), 0.93(s, 9H, tert-butyl), 0.16 (s, 3H, CH₃), 0.15 (s, 3H, CH₃); ¹³C-NMR (125MHz, CDCl₃) δ 170.9, 168.6, 138.6, 137.8, 137.7, 128.7, 128.6, 128.3,128.2, 128.2, 128.1, 127.9, 127.8, 98.0, 97.8, 83.9, 80.2, 77.6, 76.5,75.6, 75.3, 75.1, 74.9, 74.7, 69.7, 63.5, 62.4, 52.8, 25.8, 21.0, 18.2,−4.1, −5.0; FAB MS (C₄₂H₅₅N₃O₁₂Si) m/z (M)⁺ calcd 821.3555, obsd821.3549.

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate (748 mg, 0.41 mmol) and DMAP (334mg, 2.73 mmol) were dissolved in CH₂Cl₂ (5 mL) and a solution oflevulinic anhydride (390 mg, 1.82 mmol) in CH₂Cl₂ (5 ml) was added. Thereaction mixture was stirred for 2 h at room temperature andconcentrated. Flash chromatography on silica gel (hexanes:EtOAc 8:2)afforded 51 (834 mg, 0.91 mmol, quant.) as a colorless foam. [α]²⁴ _(D):+18.9 (c 1.01, CH₂Cl₂); IR (thin film on NaCl) 3036, 2929, 2857, 2108,1747, 1720, 1454, 1362 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.40-7.26 (m,15H, arom.), 5.50 (d, J=3.7 Hz, 1H, H-1A), 5.03 (dd, J=7.3, 8.8 Hz, 1H,H-2B), 4.89 (s, 2H, benzyl-CH₂), 4.83 (d, J=10.7 Hz, 2H, benzyl-CH₂),4.74 (d, J=11.3 Hz, 1H, benzyl-CH₂), 4.73 (d, J=7.3 Hz, 1H, H-1B), 4.56(d, J=11.0 Hz, 1H, benzyl-CH₂), 4.27-4.23 (m, 3H, H-4B, H-6Aa, H-6Ab),3.97 (d, J=9.5 Hz, 1H, H-5B), 3.89 (dd, J=8.8, 10.1 Hz, 1H, H-3A), 3.82(dd, J=8.9, 8.5 Hz, 1H, H-3B), 3.77 (s, 3H, OCH₃), 3.66-3.62 (m, 1H,H-5A), 3.53 (dd, J=10.1, 8.8 Hz, 1H, H-4A), 3.31 (dd, J=3.7, 10.4 Hz,1H, H-2A), 2.69-2.65 (m, 2H, Lev-CH₂), 2.54-2.49 (m, 2H, Lev-CH₂), 2.14(s, 3H, Lev-CH₃), 2.04 (s, 3H, acetyl-CH₃), 0.86 (s, 9H, tert-butyl),0.11 (s, 3H, CH₃), 0.10 (s, 3H, CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 206.2,171.3, 170.8, 168.5, 138.0, 137.7, 137.7, 128.7, 128.5, 128.2, 128.2,128.2, 128.1, 127.8, 127.7, 97.7, 96.1, 82.6, 80.2, 77.6, 75.7, 75.2,75.2, 75.2, 74.5, 74.4, 69.8, 63.5, 62.4, 52.9, 37.9, 30.0, 28.1, 25.6,21.0, 18.0, −4.2, −5.2; FAB MS (C₄₇H₆₁N₃O₁₄Si) m/z (M)⁺ calcd 919.3923,obsd 919.3914.

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl2-O-allyloxycarbonyl-3-O-benzyl-β-D-glucopyranosiduronate 52

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl-3-O-benzyl-β-D-glucopyranosiduronate(for the synthesis see the procedure to prepare compound 51), (190 mg,0.231 mmol) and DMAP (780 mg, 6.38 mmol) were dissolved in CH₂Cl₂ (5 mL)and cooled to −70° C. Allyloxycarbonylchloride (600 μL, 5.65 mmol) wasadded in three equal portions every 2 h. After the addition was completethe mixture was warmed to room temperature and stirred overnight. Themixture was poured into EtOAc and washed with 1 N HCl, brine and sat.NaHCO₃. The organic layer was dried over Na₂SO₄, filtered and thesolvent was removed under reduced pressure. Flash chromatography onsilica gel (hexanes:EtOAc 9:1→85:15) afforded 52 (190 mg, 0.21 mmol,91%) as a colorless oil. [α]²⁴ _(D): +21.9 (c 1.25, CH₂Cl₂); IR (thinfilm on NaCl) 3026, 2929, 2858, 2108, 1756, 1252 cm⁻¹; ¹H-NMR (500 MHz,CDCl₃) δ 7.41-7.26 (m, 15 H, arom.), 5.92-5.84 (m, 1H, alloc), 5.52 (d,J=3.7 Hz, 1H, H-1A), 5.36-5.32 (m, 1H, alloc), 5.26-5.23 (m, 1H, alloc),4.92-4.81 (m, 5H, 4×benzyl-CH₂, H-2B), 4.79-4.73 (m, 2H, 2×benzyl-CH ₂),4.63-4.54 (m, 3H, H-1B, alloc-CH₂), 4.29-4.26 (m, 2H, H-6Aa, H-6Ab),4.22 (dd, J=9.5, 8.9 Hz, 1H, H-4B), 3.97 (d, J=9.5 Hz, 1H, H-5B), 3.90(dd, J=8.8, 10.4 Hz, 1H, H-3A), 3.84 (dd, J=9.2, 8.8 Hz, 1H, H-3B), 3.78(s, 3H, OCH₃), 3.65-3.61 (m, 1H, H-5A), 3.54 (dd, J=8.9, 10.1 Hz, 1H,H-4A), 3.32 (dd, J=3.7, 10.4 Hz, 1H, H-2A), 2.05 (s, 3H, acetyl-CH₃),0.88 (s, 9H, tert-butyl), 0.13 (s, 3H, CH₃), 0.12 (s, 3H, CH₃); ¹³C-NMR(125 MHz, CDCl₃) δ 170.8, 168.3, 154.0, 137.9, 137.7, 137.6, 131.4,128.7, 128.7, 128.5, 128.3, 128.2, 128.2, 128.1, 127.9, 127.7, 119.6,97.8, 96.1, 82.5, 80.2, 79.1, 77.6, 75.7, 75.4, 75.2, 74.9, 74.5, 69.8,69.0, 63.5, 62.4, 52.9, 25.6, 21.0, 18.0, −4.1, −5.4; FAB MS(C₄₆H₅₉N₃O₁₄Si) m/z (M)⁺ calcd 905.3766, obsd 905.3751.

O-(6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl2-O-levulinyl-3-O-benzyl-α-D-glucopyranosiduronate trichloroacetimidate53

Compound 48 (1.06 g, 1.12 mmol) was dissolved in THF (10 mL) and cooledto 0° C. Glacial acetic acid (90 μL, 1.57 mmol) and TBAF (1M in THF,1.30 mL, 1.30 mmol) were added in sequence. After 30 min the mixture waspoured into Et₂O (100 mL) and washed with brine (3×). The organic layerwas dried over Na₂SO₄, filtered and the solvent was removed underreduced pressure. The residue was dissolved in CH₂Cl₂ (20 mL) and cooledto 0° C. Trichloroacetonitrile (1.7 mL, 17.0 mmol) and DBU (15 μL, 0.1mmol) were added and the mixture was stirred at 0° C. for 1 h and atroom temperature for 3 h. After removal of the solvent under reducedpressure, flash chromatography on silica gel (hexanes:EtOAc 85:15→70:30)afforded 53 (1.0 g, 1.03 mmol, 92%) as a colorless foam. [α]²⁴ _(D):+108.9 (c 1.69, CH₂Cl₂); IR (thin film on NaCl) 3337, 3031, 2954, 2929,2857, 2105, 1746, 1720, 1678, 1454 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 8.71(s, 1H, NH), 7.26-7.40 (m, 10H, arom.), 6.54 (d, J=3.7 Hz, 1H, H-1B),5.63 (d, J=3.7 Hz, 1H, H-1A), 5.12-5.16 (m, 1H, H-2B), 4.80-4.92 (m, 4H,4×benzyl-CH₂), 4.48 (d, J=9.5 Hz, 1H, H-5B), 4.35 (dd, J=2.1, 11.9 Hz,1H, H-6Aa), 4.21-4.29 (m, 2H), 4.04 (dd, J=4.0, 12.2 Hz, 1H, H-6Ab),3.79 (s, 3H, OCH₃), 3.69 (dd, J=8.5, 10.1 Hz, 1H), 3.64 (dd, J=9.5, 8.5Hz, 1H), 3.45-3.49 (m, 1H, H-5A), 3.23 (dd, J=4.0, 10.1 Hz, 1H, H-2A),2.61-2.71 (m, 2H, Lev-CH₂), 2.35-2.50 (m, 2H, Lev-CH₂), 2.14 (s, 3H,CH₃), 2.10 (s, 3H, CH₃), 0.88 (s, 9H, tert-butyl), 0.00 (s, 3H, CH₃),−0.01 (s, 3H, CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 206.1, 172.0, 171.0,168.7, 160.7, 138.1, 137.9, 128.7, 128.5, 128.0, 127.7, 127.6, 127.4,98.1, 93.3, 90.9, 80.1, 79.7, 75.3, 75.3, 74.1, 72.5, 72.4, 71.2, 70.9,63.7, 62.6, 53.1, 37.8, 30.0, 27.7, 26.0, 21.1, 18.1, −3.5, −4.9; FAB MS(C₄₂H₅₅Cl₃N₄O₁₄Si) m/z (M)⁺ calcd 972.2550, obsd 972.2579.

O-(6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl2-O-allyloxycarbonyl-3-O-benzyl-α-D-glucopyranosiduronatetrichloroacetimidate 54

Compound 49 (271 mg, 0.291 mmol) was dissolved in anhydrous THF (5 mL)and cooled to 0° C. Glacial acetic acid (21 μL, 0.367 mmol) and TBAF (1Min THF, 320 μL, 0.32 mmol) were added in sequence. After 30 min themixture was poured into Et₂O (50 mL) and washed with brine (3×). Theorganic layer was dried over Na₂SO₄, filtered and the solvent wasremoved under reduced pressure. The residue was dissolved in CH₂Cl₂ (11mL) and cooled to 0° C. Trichloroacetonitrile (450 μL, 4.49 mmol) andDBU (5 μL, 0.033 mmol) were added and the mixture was stirred overnightat room temperature. After removal of the solvent under reducedpressure, flash chromatography on silica gel (hexanes:EtOAc 85:15)afforded 54 (190 mg, 0.198 mmol, 68%) as a colorless foam. [α]²⁴ _(D):+114.0 (c 1.37, CH₂Cl₂); IR (thin film on NaCl) 3340, 3026, 2953, 2857,2105, 1754, 1679, 1454, 1367 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 8.74 (s,1H, NH), 7.40-7.26 (m, 10H, arom.), 6.63 (d, J=3.4 Hz, 1H, H-1B),5.91-5.82 (m, 1H, alloc), 5.65 (d, J=3.7 Hz, 1H, H-1A), 5.34-5.30 (m,1H, alloc), 5.27-5.23 (m, 1H, alloc), 5.01 (dd, J=3.4, 9.5 Hz, 1H,H-2B), 4.93-4.85 (m, 3H, 3×benzyl-CH₂), 4.82 (d, J=11.0 Hz, 1H,benzyl-CH₂) 4.65-4.57 (m, 2H, alloc), 4.50 (d, J=9.5 Hz, 1H, H-5B), 4.37(dd, J=1.8, 12.2 Hz, 1H, H-6Aa), 4.29-4.22 (m, 2H, H-3B, H-4B), 4.05(dd, J=4.0, 12.2 Hz, 1H, H-6Ab), 3.80 (s, 3H, OCH₃), 3.71 (dd, J=10.1,8.5 Hz, 1H, H-3A), 3.65 (dd, J=9.2, 8.5 Hz, 1H, H-4A), 3.50-3.46 (m, 1H,H-5A), 3.23 (dd, J=3.7, 10.1 Hz, 1H, H-2A), 2.11 (s, 3H, acetyl-CH₃),0.89 (s, 9H, tert-butyl), 0.00 (s, 3H, CH₃), −0.01 (s, 3H, CH₃); ¹³C-NMR(125 MHz, CDCl₃) δ 171.0, 168.6, 160.7, 154.2, 138.0, 137.6, 131.2,128.6, 128.4, 128.0, 127.8, 127.7, 127.4, 119.4, 98.2, 93.2, 90.8, 80.0,79.4, 75.8, 75.4, 75.2, 74.1, 72.4, 71.2, 70.9, 69.2, 63.6, 62.5, 53.0,26.0, 21.0, 18.1, −3.6, −4.9; FAB MS (C₄₁H₅₃Cl₃N₄O₁₄Si) m/z (M)⁺ calcd958.2393, obsd 958.2392

O-(6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)methyl-2-O-levulinyl-3-O-benzyl-α-D-glucopyranosiduronatetrichloroacetimidate 55

Compound 51 (345 mg, 0.376 mmol) was dissolved in THF (10 mL) and cooledto 0° C. Glacial acetic acid (27 μL, 0.472 mmol) andtetrabutylammoniumfluoride (1M in THF, 415 μL, 0.415 mmol) were added insequence. After 40 min this mixture was poured into EtOAc (50 mL) andwashed with sat. NaHCO₃. The organic layer was dried over Na₂SO₄,filtered and the solvent was removed under reduced pressure. The residuewas dissolved in CH₂Cl₂ (10 mL) and cooled to 0° C.Trichloroacetonitrile (750 μL, 7.48 mmol) and DBU (30 μL) were added andthe mixture was stirred for 2 h. After removal of the solvent underreduced pressure, flash chromatography on silica gel (hexanes:EtOAc8:2→6:4) afforded 55 as a colorless foam (302 mg, 0.32 mmol, 85%). [α]²⁴_(D): +79.8 (c 1.72, CH₂Cl₂); IR (thin film on NaCl) 3337, 3063, 3030,2953, 2108, 1746, 1719, 1677, 1497, 1363 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ8.71 (s, 1H, NH), 7.40-7.25 (m, 15H, arom.), 6.53 (d, J=3.4 Hz, 1H,H-1B) 5.55 (d, J=3.7 Hz, 1H, H-1A), 5.14-5.11 (m, 1H, H-2B), 4.95-4.82(m, 5H, benzyl-CH₂), 4.57 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.46-4.42 (m,1H, H-4B), 4.28-4.20 (m, 4H, H-3B, H-5B, H-6Aa, H-6Ab), 3.94 (dd, J=8.5,10.4 Hz, 1H, H-3A), 3.77 (s, 3H, OCH₃), 3.62-3.58 (m, 1H, H-5A), 3.51(dd, J=8.8, 10.1 Hz, 1H, H-4A) 3.30 (dd, J=3.7, 10.4 Hz, 1H, H-2A),2.70-2.60 (m, 2H, Lev-CH₂), 2.51-2.36 (m, 2H, Lev-CH₂), 2.14 (s, 3H,Lev-CH₃), 2.04 (s, 3H, acetyl-CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 206.1,172.0, 170.9, 168.6, 160.7, 138.0, 137.8, 137.7, 128.7, 128.7, 128.7,128.2, 128.1, 127.9, 127.6, 98.3, 93.2, 90.9, 80.1, 79.4, 77.6, 75.7,75.4, 75.2, 75.0, 72.5, 72.4, 70.0, 63.3, 62.4, 53.2, 37.8, 30.0, 27.7,21.0; FAB MS (C₄₃H₄₇Cl₃N₄O₁₄) m/z (M)⁺ calcd 948.2154, obsd 948.2118.

O-(6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-allyloxycarbonyl-α/β-D-glucopyranosiduronatetrichloroacetimidate 56

Compound 52 (74 mg, 0.082 mmol) was dissolved in THF (2 mL) and cooledto 0° C. Glacial acetic acid (6 μL, 0.1 mmol) and TBAF (1 M in THF, 90μL, 0.09 mmol) were added. After 30 min the mixture was poured into Et₂O(50 mL) and washed with brine (3×). The organic layer was dried overNa₂SO₄, filtered and the solvent was removed under reduced pressure. Theresidue was dissolved in CH₂Cl₂ (2 mL) and cooled to 0° C.Trichloroacetonitrile (120 μL, 1.19 mmol) and DBU (1.2 μL, 0.008 mmol)were added and the mixture was stirred overnight at room temperature.After removal of the solvent under reduced pressure, flashchromatography on silica gel (hexanes:EtOAc 85:15) afforded 56 (63 mg,0.07 mmol, 83%) as a colorless foam. FAB MS (C₄₂H₄₅Cl₃N₄O₁₄) m/z (M)⁺calcd 934.1998, obsd 934.1989.

3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl1,2-O-acetyl-3-O-benzyl-α/β-L-idopyranosiduronate 57

Pyridine (2.0 mL, 24 mmol), acetic anhydride (1.4 mL, 15 mmol) and DMAP(12 mg, 0.1 mmol) were added to a solution of 53 (700 mg, 1.02 mmol) inCH₂Cl₂ (16 mL). The solution was stirred at room temperature for 1 h,water was added and the mixture was stirred for one additional hour. Theorganic phase was washed with sat. NaHCO₃, water, and aqueous HCl (10%),dried over MgSO₄ and filtered. The solvent was removed under reducedpressure and the residue was purified by silica gel columnchromatography (hexane:EtOAc 90:20) to yield 57 (707 mg, 0.92 mmol, 95%)as a colorless syrup. FAB MS (C₃₄H₄₉N₃O₁₅Si) m/z (M⁺) calcd 767.2933,obsd 767.2951.

6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl1,2-O-acetyl-3-O-benzyl-L-idopyranosiduronate 58

Pyridine (1.5 mL, 18 mmol), acetic anhydride (1 mL, 11 mmol) and DMAP(10 mg, 0.08 mmol) were added to a solution of 42 (560 mg, 0.77 mmol) inCH₂Cl₂ (10 mL). The solution was stirred at room temperature for 1 h,water was added and the mixture was stirred for one additional hour. Theorganic phase was washed with sat. NaHCO₃, water and aqueous HCl (10%),dried over MgSO₄ and filtered. The solvent was removed under reducedpressure and the residue was purified by silica gel columnchromatography (hexane:EtOAc 90:20) to yield 58 (619 mg, 0.76 mmol, 99%)as a colorless syrup. FAB MS (C₃₉H₅₃N₃O₁₄Si) m/z (M)⁺ calcd 815.3297,obsd 815.3270.

O-3,6-Di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl2-O-acetyl-3-O-benzyl-β-L-idopyranosyluronate trichloroacetimidate 59

Benzylamine (2.0 mL, 18.3 mmol) was added to a solution of 57 (650 mg,0.85 mmol) in Et₂O (50 mL) at 0° C. After stirring at 0° C. for 4 h themixture was diluted with CH₂Cl₂, filtered and washed with HCl (10%). Theorganic phase was dried over Na₂SO₄, filtered and the solvent wasremoved under reduced pressure. Flash chromatography on silica gel(hexane:AcOEt 90:10→80:20) afforded3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl2-O-acetyl-3-O-benzyl-α/β-L-idopyranosiduronate (443 mg, 72%) as a whitesolid. FAB MS (C₃₂H₄₇N₃O₁₄Si) m/z (M)⁺ calcd 725.2827, obsd 725.2811.

A solution of3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl2-O-acetyl-3-O-benzyl-α/β-L-idopyranosiduronate (335 mg, 0.46 mmol) andtrichloroacetonitrile (1.3 mL, 12.5 mmol) in CH₂Cl₂ (10 mL) containingfreshly activated powdered 4 Å molecular sieves (100 mg) was stirred 30minutes at room temperature. After cooling the solution to 0° C. DBU (30μl, 0.2 mmol) was added. The temperature was allowed to rise and after 1h stirring, the mixture was filtered through a pad of Celite andconcentrated. The residue was purified by silica gel columnchromatography (hexane:EtOAc 85:15) yielding 59 (373 mg, 0.43 mmol, 93%)as a white solid. [α]²⁴ _(D): +53.4 (c 1.00, CHCl₃); IR (thin film onNaCl) 2935, 2693, 2109, 1744, 1675, 1372 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ8.68 (s, 1H, NH), 7.36-7.30 (m, 5H, H-arom.), 6.41 (s, 1H, H-1B), 5.26(dd, J=8.5, 10.4 Hz, 1H, H-3A), 5.17 (s, 1H, H-2B), 5.00 (d, J=3.4 Hz,1H, H-3B), 4.98 (d, J=1.8 Hz, 1H, H-1A) 4.81 (d, J=11.6 Hz, 1H,benzyl-CH₂) 4.65 (d, J=11.6 Hz, 1H, benzyl-CH₂), 4.46 (dd, J=2.1, 12.5Hz, H-6aA), 4.27 (s, 1H, H-5B), 4.09-4.06 (m, 2H, H-6bA, H-4B),4.00-3.81 (m, 1H, H-5A), 3.80 (s, 3H, OCH₃), 3.81- 3.79 (m, 1H, H-4A),3.05 (dd, J=3.3, 10.7 Hz, 1H, H-2A), 2.20 (s, 3H, COCH₃), 2.14 (s, 3H,COCH₃), 2.10 (s, 3H, COCH₃), 0.84 (s, 9H, C(CH₃)₃), 0.49 (s, 3H, SiCH₃),0.30 (s, 3H, SiCH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 170.7, 170.3, 169.9,169.1, 160.3, 137.2, 128.6, 128.1, 128.0, 98.3, 95.7, 73.7, 72.8, 72.7,72.3, 71.1, 69.2, 68.9, 65.4, 62.4, 61.9, 52.8, 25.8, 21.5, 21.0, 20.9,18.1, −4.0, −4.8; FAB MS (C₃₄H₄₇Cl₃N₄O₁₄Si) m/z (M)⁺ calc 868.1924, obsd868.1938.

O-(6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl2-O-acetyl-3-O-benzyl-β-L-idopyranosyluronate) trichloroacetimidate 60

Benzylamine (0.6 mL, 5.5 mmol) was added to a solution of 58 (600 mg,0.73 mmol) in Et₂O (15 mL) at 0° C. After stirring at 0° C. for 5 h themixture was diluted with CH₂C₂, filtered and washed with HCl (10%). Theorganic phase was dried over Na₂SO₄ and after filtration purified bysilica gel column chromatography (hexane:AcOEt 90:10→80:20) to yield6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl2-O-acetyl-3-O-benzyl-α/β-L-idopyranosiduronate (415 mg, 0.54 mmol, 77%)as a white solid. FAB MS (C₃₇H₅₁N₃O₁₃Si) m/z (M)⁺ calcd 773.3191, obsd773.3201.

A solution of6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl2-O-acetyl-3-O-benzyl-α/β-L-idopyranosiduronate (90 mg, 0.12 mmol) andtrichloroacetonitrile (0.34 mL, 3.3 mmol) in CH₂Cl₂ (3 mL) containingfreshly activated powdered 4 Å molecular sieves (50 mg) was stirred 30minutes at room temperature. After cooling the solution to 0° C. DBU (2μl, 0.012 mmol) was added. The temperature was allowed to rise and after1 h stirring, the mixture was filtered through a pad of Celite andconcentrated. The residue was purified by silica gel columnchromatography (hexane:EtOAc 85:15) yielding 60 (104 mg, 0.113 mmol,97%) as a white solid. [α]²⁴ _(D): +70.3 (c 1.00, CHCl₃); ¹H-NMR (500MHz, CDCl₃) δ 8.70 (s, 1H, NH), 7.36-7.27 (m, 10H, arom.), 6.43 (s, 1H,H-1B), 5.16 (s, 1H, H-2B), 4.93 (d, J=3.4 Hz, 1H, H-3B), 5.00 (d, J=2.1Hz, 1H, H-1A), 4.84 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.83 (d, J=11.6 Hz,1H, benzyl-CH₂), 4.76 (d, J=11.0 Hz, 1H, benzyl-CH₂), 4.67 (d, J=11.6Hz, 1H, benzyl-CH₂), 4.41 (dd, J=2.1, 12.2 Hz, H-6aA), 4.21 (s, 1H,H-5B), 3.82 (s, 3H, OCH₃), 4.07-4.02 (m, 2H, H-6bA, H-4B), 3.75-3.70 (m,1H, H-5A), 3.68-3.62 (m, 3H, H-4A, H-3A), 3.35 (dd, J=3.5, 9.7 Hz, 1H,H-2A), 2.17 (s, 3H, COCH₃), 2.07 (s, 3H, COCH₃), 0.88 (s, 9H, C(CH₃)₃),0.05 (s, 3H, SiCH₃), −0.01 (s, 3H, SiCH₃); ¹³C-NMR (125 MHz, CDCl₃) δ170.9, 170.1, 168.9, 160.3, 137.9, 137.2, 128.6, 128.5, 128.2, 127.9,127.8, 127.4, 97.1, 95.4, 80.3, 75.2, 72.6, 72.1, 71.3, 70.8, 70.8,69.3, 65.4, 64.0, 62.7, 52.7, 26.3, 21.1, 21.0, 18.1, −3.5, −4.8; FAB MS(C₃₄H₄₇Cl₃N₄O₁₄Si) m/z (M)⁺ calcd 916.2287, obsd 916.2246.

n-Pentenyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate 61

Compound 53 (292 mg, 0.30 mmol) was coevaporated with toluene (3×),dried under vacuum for 1 h, dissolved in toluene (5 mL) and4-penten-1-ol (300 μL, 3.00 mmol) was added. After cooling the mixtureto 0° C., TMSOTf (0.1 M in toluene, 300 μL, 0.03 mmol) was addeddropwise. The mixture was warmed to room temperature and stirred for 2h. Triethylamine (0.6 mL) was added and the solvent was removed underreduced pressure. Flash chromatography on silica gel (hexanes:EtOAc85:15) afforded 61 (203 mg, 0.60 mmol, 75%) as a colorless gum. [α]²⁴_(D): +53.1 (c 1.18, CH₂Cl₂); IR (thin film on NaCl) 2930, 2858, 2106,1747, 1362, 1237 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.25-7.39 (m, 10H,arom.), 5.75-5.84 (m, 1H, CH olef.), 5.54 (d, J=3.7 Hz, 1H, H-1A), 5.07(dd, J=7.3, 8.5 Hz, 1H, H-2B), 4.95-5.04 (m, 2H, olef.), 4.88 (d, J=11.0Hz, 1H, benzyl-CH₂), 4.74-4.82 (m, 3H of PhCH ₂), 4.48 (d, J=7.3 Hz, 1H,H-1B), 4.35 (dd, J=2.1, 11.9 Hz, 1H, H-6_(a)A), 4.25 ( t, J=9.2 Hz, 1H,H-4B), 4.02-4.07 (m, 2H, H-6_(b)A, H-5B), 3.84-3.89 (m, 2H, H-3B, pent),3.79 (s, 3H, COOCH₃), 3.61-3.67 (m, 2H, H-3A, H-4A), 3.43-3.51 (m, 2H,H-5A, pent), 3.23-3.26 (m, 1H, H-2A), 2.67-2.71 (m, 2H, lev-CH₂),2.44-2.58 (m, 2H, lev-CH₂), 2.14 (s, 3H, lev-CH₃), 2.03-2.12 (m, 5H,acetyl-CH₃, pent-CH₂), 1.60-1.72 (m, 2H, pent-CH₂), 0.88 (s, 9H,tert-butyl), 0.00 (s, 3H, CH₃), −0.01 (s, 3H, CH₃); ¹³C-NMR (125 MHz,CDCl₃) δ 206.7, 172.0, 171.5, 169.3, 138.7, 138.6, 138.3, 129.1, 129.0,128.4, 128.3, 128.3, 127.9, 115.7, 101.7, 98.1, 83.1, 80.7, 75.8, 75.0,75.0, 74.9, 74.1, 71.7, 71.4, 70.0, 64.4, 63.1, 53.4, 38.5, 30.6, 30.5,29.2, 28.6, 26.6, 21.6, 18.7, −3.0, −4.4; FAB MS (C₄₅H₆₃N₃O₁₄Si) m/z(M)⁺ calcd 897.4079, obsd 897.4067.

n-Pentenyl(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate 62

Compound 61 (674 mg, 0.75 mmol) was dissolved in THF (80 mL). Glacialacetic acid (20 mL) and HF/pyridine-complex (12 mL) were added and thesolution was stirred at room temperature for 93 h. The mixture waspoured into EtOAc and washed with brine, water, sat. NaHCO₃ and driedover Na₂SO₄. After filtration, the solvent was removed under reducedpressure. Flash chromatography on silica gel (hexanes:EtOAc 8:2→6:4)afforded 62 (500 mg, 0.64 mmol, 85%) as a colorless oil. [α]²⁴ _(D):+0.3 (c 1.20, CH₂Cl₂); IR (thin film on NaCl) 3484, 3037, 2924, 2109,1746, 1719, 1363 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.43-7.25 (m, 10H,arom.), 5.84-5.75 (m, 1H, CH olef.), 5.50 (d, J=3.7 Hz, 1H, H-1A), 5.07(dd, J=7.3, 8.5 Hz, 1H, H-2B), 5.04-4.95 (m, 2H, CH₂ olef.), 4.90 (d,J=11.3 Hz, 1H of PhCH ₂), 4.88 (d, J=11.3 Hz, 1H of PhCH ₂), 4.81 (d,J=10.7 Hz, 1H, PhCH ₂), 4.75 (d, J=10.7 Hz, 1H of PhCH ₂), 4.57 (dd,J=3.1, 12.5 Hz, 1H, H-6_(a)A), 4.48 (d, J=7.3 Hz, 1H, H-1B), 4.24 (dd,J=9.2 Hz, J=8.9 Hz, 1H, H-4B), 4.12 (dd, J=1.8, 12.5 Hz, 1H, H-6_(b)A),4.01 (d, J=9.5 Hz, 1H, H-5B), 3.88-3.83 (m, 2H, H-3B, pent), 3.78 (s,3H, COOCH₃), 3.74 (dd, J=8.8, 10.0 Hz, 1H, H-3A), 3.50-3.38 (m, 3H,H-4A, H-5A, pent), 3.23 (dd, J=3.7, 10.4 Hz, 1H, H-2A), 3.05 (d, J=3.4Hz, 1H, OH), 2.71-2.68 (m, 2H, lev-CH₂), 2.59-2.45 (m, 2H, lev-CH₂),2.15 (s, 3H, lev-CH₃), 2.11-2.04 (m, 5H, CH₃, pent), 1.72-1.60 (m, 2H,pent); ¹³C-NMR (125 MHz, CDCl₃) 206.2, 172.5, 171.5, 168.9, 138.1,138.0, 137.8, 128.8, 128.6, 128.4, 128.3, 127.9, 127.8, 115.2, 101.2,97.8, 82.5, 79.1, 75.5, 74.6, 74.5, 74.5, 73.7, 70.9, 70.5, 69.5, 62.9,62.6, 52.9, 38.0, 30.1, 30.0, 28.7, 28.1, 21.0; FAB MS (C₃₉H₄₉N₃O₁₄) m/z(M)⁺ calcd 783.3215, obsd 783.3206.

n-Pentenyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-β-O-D-glucopyranosiduronate 63

Compound 46 (816 mg, 0.856 mmol) was coevaporated with toluene (3×),dried under vacuum for 1 h, dissolved in toluene (30 mL) and4-penten-1-ol (450 μL, 4.36 mmol) was added. After cooling the mixtureto 0° C., TMSOTf (0.1 M in toluene, 1.72 mL, 0.17 mmol) was addeddropwise. The mixture was warmed to room temperature and stirred for 48h. Triethylamine (1.7 mL) was added and the solvent was removed underreduced pressure. Flash chromatography on silica gel (hexanes:EtOAc85:15) afforded 63 (527 mg, 0.60 mmol, 70%) as a colorless gum. [α]²⁴_(D): +45.5 (c 1.00, CHCl₃); IR (thin film on NaCl) 2954, 2109, 1745,1223, 1072, 838 cm⁻¹;¹H-NMR (500 MHz, CDCl₃) δ 7.37-7.28 (m, 10H,H-arom.), 5.79-5.74 (m, 1H, CH olef.), 5.50 (d, J=3.7 Hz, 1H, H-1A),5.09 (dd, J=7.6, 8.8 Hz, H-2B), 5.07-4.95 (m, 2H, CH₂ olef.), 4.74 (d,J=7.3 Hz, H-1B), 4.87 (d, J=11.3 Hz, 2H, PhCH ₂), 4.86 (d, J=11.0 Hz,2H, PhCH ₂), 4.80 (d, J=11.3 Hz, 1H, PhCH ₂),4.70 (d, J=11.0 Hz, 1H,PhCH ₂), 4.48 (d, 1H, H-1B), 4.35 (dd, J=2.1, 11.9 Hz, 1H, H-6_(a)A),4.04 (t, 1H, H-4B), 4.05-4.00 (m, 2H, H-5B, H-6_(b)A), 3.89-3.80 (m, 4H,H-3B, CH₂Cl, 1H of OCH₂), 3.79 (s, 3H, OCH₃), 3.65-3.64 (m, 2H, H-3A,H-4A) 3.50-3.42 (m, 2H, H-5A, 1H of OCH₂), 3.28 (dd, J=4.0, 10.1 Hz, 1H,H-2A), 2.09 (s, 3H, COCH₃), 1.07-1.03 (m, 2H pent-CH₂), 1.69-1.58 (m, 2Hpent-CH₂), 0.88 (s, 9H, tert-butyl), −0.01 (s, 6H, 2 CH₃); ¹³C-NMR (125MHz, CDCl₃) δ 171.0, 168.6, 166.0, 137.9, 137.7, 137.4, 129.0, 128.7,128.5, 128.1, 128.0, 127.9, 127.75, 127.7, 127.4, 115.0, 100.9, 97.7,82.5, 80.2, 75.3, 74.9, 74.8, 74.6, 74.5, 71.3, 70.8, 69.5, 63.8, 62.5,52.9, 44.0, 42.8, 40.6, 30.0, 28.6, 26.0, 21.0, 18.1, −3.5, −4.9; FAB MS(C₄₂H₃₆ClN₃O₁₃Si) m/z (M)⁺ calcd 875.3427, obsd 875.3432.

n-Pentenyl(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-β-O-pentenyl-D-glucopyranosiduronate 64

Compound 63 (250 mg, 0.285 mmol) was dissolved in THF (33 mL). Glacialacetic acid (8 mL) and HF/pyridine-complex (4.8 mL) were added and thesolution was stirred at room temperature for 4 days. The mixture waspoured into Et₂ O and washed with brine, sat. NaHCO₃ and dried overNa₂SO₄. After filtration, the solvent was removed under reducedpressure,. Flash chromatography on silica gel (hexanes:EtOAc 7:3)afforded 64 (186 mg, 0.244 mmol, 85%) as a colorless foam. [α]²⁴ _(D):+10.8 (c1.00, CHCl₃); IR (thin film on NaCl) 3485, 2925, 2109, 1747,1454, 1028 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.43-7.27 (m, 10H, arom.),5.98-5.70 (m, 1H, CH olef.), 5.47 (d, J=3.8 Hz, H-1A), 5.09 (t, 1H,H-2B), 5.08-4.97 (m, 2H, CH₂ olef.), 4.95-4.93 (m, 3H, PhCH ₂), 4.69 (d,J=10.7 Hz, 1H of PhCH ₂) 4.40-4.35 (m, 1H, H-2B), 4.59 (dd, J=2.7, 12.4Hz, 1H, H-6_(a)A), 4.48 (d, J=7.7 Hz, H-1B), 4.24 (t, 1H, H-4B), 4.11(dd, J=1.9, 12.4 Hz, 1H, H-6_(b)A), 4.0 (d, 1H, J=5.5 Hz, H-5B),3.89-3.79 (m, 4H, H-3B, CH₂Cl, 1H of OCH₂), 3.79 (s, 3H, COOCH₃),3.78-3.70 (m, 1H, H-3A), 3.50-3.38 (m, 3H, H-4A, H-5A, 1H of OCH₂), 3.27(dd, J=3.8, 10.4 Hz, 1H, H-2A), 2.98 (bs, 1H, OH), 2.03-2.01 (m, 2H,pent-CH₂), 2.12 (s, 3H, CH₃), 1.73-1.59 (m, 2H, pent-CH₂); ¹³C-NMR (125MHz, CDCl₃) δ 172.5, 168.6, 165.9, 138.0, 137.9, 137.7, 128.8, 128.7,128.4, 128.3, 128.1, 127.7, 115.3, 101.0, 98.0, 82.5, 79.0, 75.5, 75.1,75.0, 74.8, 74.6, 71.1, 70.5, 69.6, 62.9, 62.6, 53.0, 40.6, 30.0, 28.6,21.0; FAB MS (C₃₆H₄₄ClN₃O₁₃) m/z (M)⁺ calcd 761.2563, obsd 761.2557.

n-Pentenyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl2-O-allyloxycarbonyl-3-O-benzyl-β-D-glucopyranosiduronate 65

Compound 54 (93 mg, 0.097 mmol) was coevaporated with toluene (3×),dried under vacuum for 1 h, dissolved in toluene (5 mL) and4-penten-1-ol (50 μL, 0.484 mmol) was added. After cooling the mixtureto 0° C., TMSOTf (0.1 M in toluene, 200 μL, 0.019 mmol) was addeddropwise. The mixture was warmed to room temperature and stirred for 30min. Triethylamine (200 μL) was added and the solvent was removed underreduced pressure. Flash chromatography on silica gel (hexanes:EtOAc85:15) afforded 65 (57 mg, 0.064 mmol, 66%) as a colorless gum. [α]²⁴_(D): +64.1 (c 1.65, CH₂Cl₂); IR (thin film on NaCl) 3065, 2953, 2857,2106, 1755, 1454, 1369 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.26-7.39 (m,10H, arom.), 5.73-5.92 (m, 2H, alloc, pent), 5.55 (d, J=3.7 Hz, 1H,H-1A), 5.32-5.37 (m, 1H, alloc), 5.19-5.25 (m, 1H, alloc), 4.96-5.04 (m,2H, pent), 4.76-4.90 (m, 5H, H-2B, 2H of PhCH ₂), 4.55-4.64 (m, 2H,alloc), 4.49 (d, J=7.6 Hz, 1H, H-1B), 4.35 (dd, J=2.1, 11.9 Hz, 1H,H-6_(a)A), 4.21 (at, J=9.2 Hz, 1H, H-4B), 4.05 (dd, J=4.0, 12.2 Hz, 1H,H-6_(b)A), 4.00 (d, J=9.5 Hz, 1H, H-5B), 3.86-3.95 (m, 2H, H-3B, pent),3.80 (s, 3H, COOCH₃), 3.62-3.70 (m, 2H, H-3A, H-4A), 3.45-3.50 (m, 2H,H-5A, pent), 3.29-3.24 (m, 1H, H-2A), 2.00-2.16 (m, 5H, CH₃, pent-CH₂),1.60-1.77 (m, 2H, pent-CH₂), 0.89 (s, 9H, tert-butyl), 0.01 (s, 3H,CH₃), 0.00 (s, 3H, CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 171.0, 168.6, 154.0,138.1, 138.0, 137.7, 131.4, 128.6, 128.5, 128.0, 127.8, 127.7, 127.4,119.6, 115.2, 101.2, 97.8, 82.6, 80.2, 77.4, 75.3, 74.9, 74.7, 74.5,71.2, 70.9, 69.7, 69.1, 63.9, 62.5, 52.9, 30.0, 28.7, 26.0, 21.1, 18.1,−3.5, −4.9; FAB MS (C₄₄H₆₁N₃O₁₄Si) m/z (M)⁺ calcd 883.3923, obsd883.3930.

n-Pentenyl(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl2-O-allyloxycarbonyl-3-O-benzyl-β-D-glucopyranosiduronate 66

Compound 65 (55 mg, 0.062 mmol) was dissolved in THF (7 mL). Glacialacetic acid (1.75 mL) and HF/pyridine-complex (1 mL) were added and thesolution was stirred at room temperature for 5 days. The mixture waspoured into EtOAc and washed with brine, water, sat. NaHCO₃ and driedover Na₂SO₄. After filtration, the solvent was removed under reducedpressure. Flash chromatography on silica gel (hexanes:EtOAc 7:3)afforded 66 (41 mg, 0.053 mmol, 85%) as a colorless oil. [α]²⁴ _(D):+11.3 (c 1.02, CH₂Cl₂); IR (thin film on NaCl) 3470, 2922, 2109, 1752,1454, 1367, 1255 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ 7.27-7.43 (m, 10H,arom.), 5.84-5.92 (m, 1H, alloc), 5.74-5.82 (m, 1H, pent), 5.51 (d,J=3.7 Hz, 1H, H-1A), 5.31-5.37 (m, 1H, alloc), 5.23-5.26 (m, 1H, alloc),4.95-5.04 (m, 2H, CH₂ olef.), 4.79-4.90 (m, 4H, H-2B, 3H of PhCH ₂),4.77 (d, J=10.7 Hz, 1H of PhCH ₂) 4.56-4.65 (m, 3H, alloc, H-6_(a)A),4.49 (d, J=7.7 Hz, 1H, H-1B), 4.21 (dd, J=8.9, 9.4 Hz, 1H, H-4B), 4.11(dd, J=1.9, 12.6 Hz, 1H, H-6_(b)A), 3.99 (d, J=9.5 Hz, 1H, H-5B),3.87-3.92 (m, 1H; pent), 3.86 (at, J=9.0 Hz, 1H, H-3B), 3.79 (s, 3H,COOCH₃), 3.74 (dd, J=8.6, 10.3 Hz, 1H, H-3A), 3.45-3.51 (m, 2H, pent,H-5A), 3.41 (dd, J=10.0, 8.7 Hz, 1H, H-4), 3.25 (dd, J=3.7, 10.4 Hz, 1H,H-2A), 2.98 (bs, 1H, OH), 2.06-2.21 (m, 5H, pent, CH₃), 1.60-1.74 (m,2H, pent); ¹³C-NMR (125 MHz, CDCl₃) δ 172.5, 168.7, 154.0, 138.1, 138.0,137.7, 131.4, 128.8, 128.6, 128.4, 128.3, 128.0, 127.8, 119.6, 115.2,101.3, 98.0, 82.6, 79.1, 75.6, 75.1, 74.9, 74.5, 71.0, 70.5, 69.7, 69.1,62.9, 62.6, 53.0, 30.0, 28.7, 21.0; FAB MS (C₃₈H₄₇N₃O₁₄) m/z (M)⁺ calcd769.3058, obsd 769.3051.

n-Pentenyl(3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-idopyranosyluronate)-(1→4)-(6-O-acetyl-3-O-benzyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-β-D-glucopyranosiduronate 67

Compound 59 (360 mg, 0.41 mmol) and 64 (186 mg, 0.24 mmol) werecoevaporated with toluene and dried under vacuum for 1 h. The mixturewas dissolved in CH₂Cl₂ (3 mL) and after cooling to −25° C., TMSOTf (370μL, 0.1M in CH₂Cl₂) was added. The mixture was stirred for 4 h and thendiluted with CH₂Cl₂ and filtered through a pad of Celite. The solventwas removed under reduced pressure and the residue was purified bysilica gel column chromatography (toluene:EtOAc 90:10→80:20) to yield 67(331 mg, 0.22 mmol, 91%) as a syrup. [α]²⁴ _(D): +40.0 (c 1.00, CHCl₃);IR (thin film on NaCl) 2930, 2107, 1728, 1538, 1362 cm⁻¹; ¹H-NMR (500MHz, CDCl₃) δ 7.39-7.27 (m, 20H, H-arom.), 5.77-5.74 (m, 1H, CH olef.),5.43 (d, J=3.7 Hz 1H, H-1C), 5.35 (d, J=4.9 Hz, 1H, H-1B), 5.23 (t,J=10.4 Hz, 1H, H-3A), 5.12 (d, J=3.7 Hz, 1H, H-1A), 5.07 (t, 1H, H-2D),5.01-4.84 (m, 6H, H-2B, CH₂ olef, PhCH ₂, H-6_(a)A), 4.73-4.64 (m, 4H, 2PhCH ₂), 4.57 (d, J=4.6 Hz, 1H, H-5B), 4.47 (d, J=7.6 Hz, H-1D), 4.34(m, 2H, H-6_(a)A,H-6_(a)C), 4.24-4.18 (m, 2H, H-6_(b)C, H-4D), 4.09 (t,1H, H-4B), 4.05 (dd, 1H, J=3.3, 12.2 Hz, H-6bA), 4.00 (m, 2H, H-5D,H-3B), 3.92-3.81 (m, 6H, 1H of OCH₂, CH₂Cl, H-5A, H-3D, H-4C), 3.79-3.72(m, 2H, H-4A, H-3C), 3.69 (s, 3H, COOCH₃), 3.67 (s, 3H, COOCH₃),3.56-3.54 (m, 1H, H-5C), 3.45-3.42 (m, 1H, 1H of OCH₂), 3.31 (dd, J=3.7,10.7 Hz, 1H, H-2C), 3.00 (dd, J=3.7, 10.7 Hz, 1H, H-2A), 2.13 (s, 6H, 2CH₃), 2.10 (s, 3H, CH₃), 2.05-2.01 (m, 2H, pent-CH₂), 2.03 (s, 3H, CH₃),1.69-1.60 (m, 2H, pent-CH₂), 0.84 (s, 9H, tert-butyl), 0.04 (s, 3H,CH₃), 0.02 (s, 3H, SiCH₃); ¹³C-NMR (125 MHz, CDCl₃) δ 171.0, 170.7,170.2, 169.8, 168.6, 165.9, 137.9, 137.7, 137.5, 128.7, 128.4, 128.2,128.1, 128.0, 127.9, 127,7, 127.3, 115.3, 101.0, 98.2, 97.7, 82.5, 78.2,76.0, 75.1, 75.0, 74.7, 74.6, 74.4, 73.0, 72.6, 71.0, 69.8, 69.6, 69.0,63.3, 62.4, 61.6, 52.4, 40.7, 30.0, 28.7, 26.6, 25.7, 21.8, 21.2, 18.0,−3.9, −4.8; FAB MS (C₆₈H₈₉ClN₆O₂₆Si) m/z (M)⁺ calcd 1468.5284, obsd1468.5361.

n-Pentenyl(3,6-di-O-acetyl-2-azido-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-β-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate 68

Compound 62 (290 mg, 0.33 mmol) and 5 (173 mg, 0.22 mmol) werecoevaporated with toluene and dried under vacuum for 1 h. The mixturewas dissolved in CH₂Cl₂ (3 mL) and after cooling to −25° C., TMSOTf (330μL, 0.1 M in CH₂Cl₂) was added. The mixture was stirred for 4 h and thendiluted with CH₂Cl₂ and filtered through a pad of Celite. The solventwas removed under reduced pressure and the residue was purified bysilica gel column chromatography (toluene:EtOAc 90:10→80:20) to yield 68(289 mg, 88%) as a syrup. ¹H-NMR (500 MHz, CDCl₃) δ 7.37-7.15 (m, 15H),5.83-5.75 (m, 1H), 5.47 (d, J=4.0 Hz, 1H), 5.35 (d, J=4.9 Hz, 1H), 5.24(dd, J=8.8, 10.7 Hz, 1H), 5.13 (d, J=3.3 Hz, 1H), 5.08-5.03 (m, 1H),4.99-4.90 (m, 4H), 4.80-4.67 (m, 5H), 4.58 (d, J=4.9 Hz, 1H), 4.47 (d,J=7.3 Hz, 1H), 4.37-4.31 (m, 2H), 4.25-4.18 (m, 2H), 4.10 (at, J=6.1 Hz,1H), 4.06 (dd, J=3.7, 12.2 Hz, 1H), 4.02-3.98 (m, 2H), 3.94-3.91 (m,1H), 3.88-3.80 (m, 3H), 3.79-3.69 (m, 2H), 3.68 (s, 3H), 3.65 (s, 3H),3.56-3.54 (m, 1H), 3.48-3.43 (m, 1H), 3.28 (dd, J=6.9, 10.3 Hz, 1H),3.00 (dd, J=3.3, 10.7 Hz, 1H), 2.68 (t, J=6.7 Hz, 1H), 2.57-2.44 (m,2H), 2.26-2.01 (m, 17H), 1.71-1.61 (m, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ206.2, 171.4, 170.6, 170.2, 170.0, 169.8, 168.9, 138.1, 138.0, 137.8,137.5, 129.2, 128.7, 128.6, 128.4, 128.2, 128.1, 127.9, 127.7, 125.5,115.1, 101.2, 98.3, 98.2, 97.3, 82.5, 78.1, 76.2, 76.0, 75.1, 74.5,74.4, 74.3, 73.5, 73.0, 72.5, 70.9, 70.4, 70.3, 69.6, 69.4, 68.9, 63.2,62.4, 61.8, 61.5, 52.8, 52.3, 37.9, 30.0, 29.9, 28.7, 28.0, 25.7, 21.6,21.5, 21.0, 20.9, 18.0, −3.9, −4.9.

n-Pentenyl(6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-O-D-glucopyranosiduronate 69

Compound 60 (85 mg, 0.09 mmol) and 62 (60 mg, 0.07 mmol) werecoevaporated with toluene and dried under vacuum for 1 h. The mixturewas dissolved in CH₂Cl₂ (2 mL) and after cooling to −25° C., TMSOTf (90μL, 0.1 M in CH₂Cl₂) was added. The mixture was stirred for 4 h and thendiluted with CH₂Cl₂ and filtered through a pad of Celite. The solventwas removed under reduced pressure and the residue was purified bysilica gel column chromatography (toluene:EtOAc 90:10→80:20) to yield 69(107 mg, 86%) as a syrup. ¹H-NMR (500 MHz, CDCl₃) δ 7.37-7.25 (m, 20H),5.82-5.74 (m, 1H), 5.47 (d, J=3.6 Hz, 1H), 5.26 (d, J=4.6 Hz, 1H),5.0.7-5.02 (m, 3H), 4.99-4.88 (m, 3H), 4.83-4.65 (m, 8H), 4.47 (d, J=7.3Hz, 1H), 4.35-4.32 (m, 2H), 4.25-4.18 (m, 2H), 4.07-3.95 (m, 4H),3.87-3.82 (m, 3H), 3.80-3.73 (m, 2H), 3.72-3.64 (m, 4H), 3.61 (s, 3H),3.59-3.55 (m, 2H), 3.47-3.43 (m, 1H), 3.27 (dd, J=3.9, 10.3 Hz, 1H),3.23 (dd, J=3.3, 10.1, 1H), 2.68 (t, J=6.7 Hz, 2H), 2.57-2.44 (m, 2H),2.18 (s, 3H), 2.14 (s, 3H), 2.13-2.05 (m, 5H), 2.02 (s, 3H), 1.70-1.61(m, 2H), 0.89 (m, 9H), −0.01 (s, 3H), −0.02 (s, 3H); ¹³C-NMR (125 MHz,CDCl₃) δ 206.2, 171.4, 170.9, 170.8, 170.1, 169.6, 168.9, 138.1, 137.9,137.8, 137.7, 137.5, 128.7, 128.6, 128.4, 128.2, 128.0, 127.9, 127.8,127.7, 127.4, 115.2, 101.2, 98.2, 97.7, 97.4, 82.6, 80.2, 78.1, 75.9,75.2, 75.0, 74.8, 74.5, 74.0, 73.6, 72.7, 71.2, 70.8, 70.2, 70.0, 69.7,69.5, 63.7, 63.2, 62.6, 61.9, 52.9, 52.1, 38.0, 30.1, 30.0, 28.7, 28.0,26.0, 21.1, 21.0, 18.1, −3.53, −4.87.

n-Pentenyl(3,6-di-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-monochloroacetyl-β-D-glucopyranosiduronate 70

Compound 67 (60 mg, 0.21 mmol) was dissolved in THF (26 mL). Glacialacetic acid (6.4 mL) and HF/pyridine-complex (3.8 mL) were added and thesolution was stirred at room temperature for three days. The mixture waspoured into EtOAc and washed with brine, water, sat. NaHCO₃ and driedover Na₂SO₄. After filtration, the solvent was removed under reducedpressure. Flash chromatography on silica gel (hexanes/EtOAc 5:3)afforded 70 (247 mg, 85%). [α]²⁴ _(D): +20.4 (c 1.00, CHCl₃); IR (thinfilm on NaCl) 3510, 2924, 2109, 1742 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ7.39-7.27 (m, 20H, arom.), 5.80-5.75 (m, 1H, CH olef.), 5.44 (d, J=3.7Hz 1H, H-1C), 5.31 (d, J=4.9 Hz, 1H, H-1B), 5.22 (t, J=10.2 Hz, 1H,H-3A), 5.16-5.12 (m, 2H), 5.07-4.83 (m, 5H), 4.73-4.64 (m, 5H),4.57-4.52 (d, 1H), 4.37 (d, 1H), 4.34-4.25 (m, 3H), 4.08 (m, 1H),4.00-3.95 (m, 3H), 3.90-3.81 (m, 6H), 3.80-3.72 (m, 2H), 3.69 (s, 3H,COOCH₃), 3.67 (s, 3H, COOCH₃), 3.60-3.53 (m, 1H, H-5C), 3.51-3.42 (m, 1Hof OCH₂), 3.27 (dd, J=3.7, 10.7 Hz, 1H, H-2C), 3.17 (dd, J=3.7, 10.7 Hz,1H, H-2A), 3.07 (bs, 1H, OH), 2.14 (s, 3H, CH₃), 2.13 (s, 3H, CH₃), 2.12(s, 3H, CH₃), 2.05-2.01 (m, 2H, pent-CH₂), 2.08 (s, 3H, CH₃), 1.74-1.60(m, 2H, pent-CH₂); ¹³C-NMR (125 MHz, CDCl₃) δ 171.9, 171.3, 171.1,170.3, 169.7, 168.6, 165.9, 138.0, 137.7, 137.4, 115.3, 100.9, 98.6,98.4, 97.6, 82.5, 78.2, 75.8, 75.7, 75.1, 75.0, 74.9, 74.7, 74.6, 74.0,73.8, 72.4, 71.4, 70.2, 70.1, 69.8, 69.5, 68.9, 63.3, 62.5, 61.9, 61.1,52.9, 52.4, 40.6, 30.0, 28.7, 21.0, 21.0, 20.9; FAB MS (C₆₂H₇₅ClN₆O₂₆)m/z (M)⁺ calcd 1354.4420, obsd 1354.441.

n-Pentenyl(3,6-di-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate 71

Compound 68 (170 mg, 0.11 mmol) was dissolved in THF (13 mL). Glacialacetic acid (3 mL) and HF/pyridine-complex (1.88 mL) were added and thesolution was stirred at room temperature for three days. The mixture waspoured into EtOAc and washed with brine, water, sat. NaHCO₃ and driedover Na₂SO₄. After filtration, the solvent was removed under reducedpressure. Flash chromatography on silica gel (hexanes:EtOAc 5:3)afforded 71 (135 mg, 86%) as a pale yellow oil. ¹H-NMR (500 MHz, CDCl₃)δ 7.37-7.15 (m, 15H), 5.82-5.74 (m, 1H), 5.47 (d, J=3.6 Hz, 1H), 5.29(d, J=4.3 Hz, 1H), 5.21 (t, J=10.1 Hz, 1H), 5.07-5.02 (m, 2H), 4.98-4.90(m, 3H), 4.79-4.68 (m, 5H), 4.61 (d, J=4.3 Hz, 1H), 4.50-4.46 (m, 2H),4.32-4.30 (m, 1H), 4.25-4.14 (m, 3H), 4.07 (t, J=5.2 Hz, 1H), 3.98 (d,J=9.5 Hz, 1H), 3.95 (t, J=5.5 Hz, 1H), 3.90-3.82 (m, 4H), 3.74 (t,J=10.1 Hz, 1H), 3.67 (s, 3H), 3.66 (s, 3H), 3.56-3.54 (m, 1H), 3.47-3.42(m, 2H), 3.27 (dd, J=3.6, 10.3 Hz, 1H), 3.17 (dd, J=3.6, 10.7 Hz, 1H),3.01 (bs, 1H), 2.68 (t, J=6.7 Hz, 2H), 2.57-2.43 (m, 2H), 2.26-2.08 (m,17H), 1.70-1.60 (m, 2H).

n-Pentenyl(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-O-pentenyl-D-glucopyranosiduronate 72

Compound 69 (70 mg, 0.045 mmol) was dissolved in THF (5.3 mL). Glacialacetic acid (1.3 mL) and HF/pyridine-complex (0.8 mL) were added and thesolution was stirred at room temperature for three days. The mixture waspoured into EtOAc and washed with brine, water, sat. NaHCO₃ and driedover Na₂SO₄. After filtration, the solvent was removed under reducedpressure. Flash chromatography on silica gel (hexanes/EtOAc 5:3)afforded 72 (50 mg, 75%). ¹H-NMR (500 MHz, CDCl₃) δ 7.40-7.25 (m, 20H),5.82-5.76 (m, 1H), 5.48 (d, J=3.6 Hz, 1H), 5.25 (d, J=4.6 Hz, 1H),5.07-4.89 (m, 6H), 4.84 (s, 2H), 4.80-4.68 (m, 5H), 4.63 (d, J=4.3 Hz,1H), 4.51 (dd, J=3.5, 12.5 Hz, 1H), 4.47 (d, J=7.3 Hz, 1H), 4.34-4.32(m, 1H), 4.26-4.20 (m, 2H), 4.09 (dd, J=2.1, 12.5, 1H), 4.04 (t, J=5.5Hz, 1H), 4.00 (d, J=9.5 Hz, 1H), 3.95 (t, J=5.5 Hz, 1H), 3.88-3.82 (m,3H), 3.78-3.71 (m, 3H), 3.69-3.64 (m, 4H), 3.57 (s, 3H), 3.48-3.43 (m,2H), 3.27 (d, J=3.6, 10.1 Hz, 1H), 3.21 (d, J=3.4, 10.1 Hz, 1H), 2.91(d, J=4.0 Hz, 1H), 2.68 (t, J=6.7 Hz, 2H), 2.56-2.45 (m, 2H), 2.14 (s,6H), 2.12-2.05 (m, 8H), 1.69-1.62 (m, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ206.2, 172.1, 171.4, 171.0, 170.0, 169.6, 168.9, 138.1, 138.0, 137.9,137.5, 129.2, 129.0, 128.8, 128.7, 128.6, 128.4, 128.4, 128.2, 128.1,128.0, 127.9, 127.8, 115.1, 101.2, 98.4, 98.2, 97.4, 82.5, 78.8, 78.2,77.4, 75.7, 75.3, 75.2, 75.0, 74.5, 74.4, 73.9, 73.6, 73.4, 71.1, 70.5,70.2, 70.0, 69.7, 69.4, 63.2, 62.8, 62.7, 61.9, 52.8, 52.2, 37.9, 30.0,28.7, 28.0, 21.1, 20.9.

O-(Methyl2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-β-L-idopyranosiduronate)trichloroacetimidate 73

tert-Butyldimethylsilyl trifluoromethanesulfonate (448 μL, 1.95 mmol)was added under argon to a solution of methyl3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranosiduronate (600 mg, 1.77mmol) and 2,6-lutidine (522 μL, 4.48 mmol) in CH₂Cl₂ (4 mL). Afterstirring for 1 h at room temperature, the reaction mixture was quenchedwith the addition of sat NaHCO₃. The mixture was diluted with CH₂Cl₂,the two phases were separated and the aqueous phase was extracted withCH₂Cl₂ (4×). The combined organic phases were dried over MgSO₄ and afterfiltration the solvent was removed under reduced pressure. Flashchromatography on silica gel (Hexanes:EtOAc 20:1) afforded methyl3-O-benzyl-4-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-β-L-idopyranosiduronate(786 mg, 98%) as a colorless solid. ¹H-NMR (500 MHz, CDCl₃) δ 7.39-7.32(m, 5H, arom.), 5.32 (d, J=2.4 Hz, 1H, H-1), 4.68 (d, J=11.9 Hz, 1H ofPhCH ₂), 4.62 (d, J=11.9 Hz, 1H of PhCH ₂), 4.38 (d, J=1.2 Hz, 1H, H-5),4.06 (m, 1H, H-3), 3.94 (bs, 1H, H-4), 3.82 (m, 1H, H-2), 3.76 (s, 3H,COOCH₃), 1.59 (s, 3H, CH₃), 1.38 (s, 3H, CH₃), 0.82 (s, 9H, tert-butyl),−0.06 (s, 3H, CH₃), −0.07 (s, 3H, CH₃); ¹³C-NMR (125 MHz, CDCl₃) δ169.8, 137.5, 128.8, 128.4, 128.1, 112.3, 96.9, 75.3, 75.1, 72.9, 72.7,68.0, 52.3, 28.3 26.6, 25.6, 18.0, −4.4, −5.3; FAB MS (C₂₃H₃₆O₇Si) m/z(M⁺) calcd 452.2230, obsd 452.2211.

A solution of methyl3-O-benzyl-4-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-β-L-idopyranosiduronate(800 mg, 1.77 mmol) in dichloroacetic acid (40 mL, 60% aq) was stirredat room temperature for 3 h and diluted with water and neutralized withNaHCO₃ (24 g). The aqueous phase was washed three times with CH₂Cl₂ andthe combined organic phases were dried over MgSO₄. After filtration, thesolvent was removed under reduced pressure to afford methyl3-O-benzyl-4-O-tert-butyldimethylsilyl-L-idopyranosiduronate (671 mg,1.62 mmol, 92%) as a white solid. The compound can be further purifiedby silica gel column chromatography (hexane:EtOAc 70:30). FAB MS(C₂₀H₃₂O₇Si) m/z (M⁺) calcd 412.1917, obsd 412.1896.

Pyridine (3.0 mL, 36 mmol), acetic anhydride (2.0 mL, 21.7 mmol) andDMAP (17 mg, 0.145 mmol) were added to a solution of3-O-benzyl-4-O-tert-butyldimethylsilyl-L-idopyranosiduronate (600 mg,1.45 mmol) in CH₂Cl₂ (20 mL). The solution was stirred at roomtemperature for 6 h, water was added and the mixture was stirred for oneadditional hour. The organic phase was washed with saturated solution ofNaHCO₃, water and 10% HCl, dried over MgSO₄ and filtered. The solventwas removed under reduced pressure and the residue was purified bysilica gel column chromatography (hexane:EtOAc 90:20) to yield methyl1,2-di-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-α/β-L-idopyranosiduronate(708 mg, 1.42 mmol, 98%) as a colorless syrup. FAB MS (C₂₄H₃₆O₉Si) m/z(M)⁺ calcd 496.2129, obsd 496.2129.

Benzylamine (600 μL, 5.4 mmol) was added to a solution of methyl1,2-di-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-α/β-L-idopyranosiduronate(630 mg, 1.27 mmol) in Et₂O (40 mL) at 0° C. After 6 h, the mixture wasdiluted with CH₂Cl₂, filtered and washed with aqueous HCl (10%). Theorganic phase was dried over MgSO₄ and after filtration silica gelcolumn chromatography (hexanes:EtOAc 90:10→80:20) afforded methyl2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-L-idopyranosiduronate(432 mg, 0.95 mmol, 75%) as a white solid. FAB MS (C₂₂H₃₄O₈Si) m/z (M)⁺calcd 454.2023, obsd 454.2016.

A solution of methyl2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-L-idopyranosiduronate(500 mg, 1.10 mmol) in CH₂Cl₂ (25 mL) was cooled to 0° C.Trichloroacetonitrile (1.7 mL, 17.0 mmol) and DBU (25 μL, 0.16 mmol)were added and after stirring the mixture at 0° C. for 1 h the solventswere removed under reduced pressure. Flash chromatography on silica gel(hexanes:EtOAc 85:15→70:30) afforded 73 (606 mg, 92%) as a colorlessfoam. ¹H-NMR (500 MHz, CDCl₃) δ 8.65 (s, 1H, NH), 7.38-7.31 (m, 5H,arom.), 6.41 (s, 1H, H-1), 5.11 (m, 1H, H-2), 4.90 (d, J=1.8 Hz, 1H,H-5), 4.80 (d, J=11.9 Hz, 1H of PhCH ₂), 4.61 (d, J=11.9 Hz, 1H of PhCH₂), 4.12 (m, 1H, H-3), 3.78 (s, 3H, COOCH₃), 3.66 (bs, 1H, H-4), 1.59(s, 3H, CH₃), 1.38 (s, 3H, CH₃), 2.08 (s, 3H, CH₃), 0.83 (s, 9H,tert-butyl), −0.05 (s, 3H, CH₃), −0.13 (s, 3H, CH₃); ¹³C-NMR (125 MHz,CDCl₃) δ 170.7, 170.0, 160.8, 138.0, 129.7, 129.1, 128.9, (128.7×2),96.1, 74.2, 72.6, 71.3, 68.8, 66.1, 53.0, (26.2), 26.1, 21.7, 1.8.5,−3.9, −4.9; FAB MS (C₂₄H₃₄Cl₃NO₈Si) m/z (M)⁺ calcd 597.119, obsd597.1143.

n-Pentenyl(methyl2-O-acetyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-α-L-idopyranosiduronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosid-uronate 74

Compound 73 (204 mg, 0.34 mmol) and 62 (206 mg, 0.26 mmol) werecoevaporated with toluene and dried under vacuum for 1 h. The mixturewas dissolved in CH₂Cl₂ (3 mL) and after cooling to −25° C., TMSOTf (340μL, 0.1M in CH₂Cl₂) was added. The mixture was stirred for 4 h and thendiluted with CH₂Cl₂ and filtered. The solvent was removed under reducedpressure and the residue was purified by silica gel columnchromatography (toluene:EtOAc 90:10→80:20) to yield 74 (298 mg, 0.24mmol, 93%) as a syrup. ¹H-NMR (500 MHz, CDCl₃) δ 7.39-7.27 (m, 15H),5.80-5.77 (m, 1H), 5.44 (d, J=3.7 Hz, 1H), 5.32 (d, J=5.8 Hz, 1H),5.07-5.02 (m, 2H), 4.98-4.95 (m, 2H), 4.90-4.87 (m, 1H), 4.79 (d, J=10.9Hz, 1H), 4.73-4.66 (m, 4H), 4.49 (d, J=5.2, 1H), 4.46 (d, J=7.3 Hz, 1H),4.38 (dd, 1H), 4.21-4.12 (m, 2H), 3.99-3.91 (m, 2H), 3.89-3.81 (m, 3H),3.75 (m, 2H), 3.71 (s, 3H), 3.68 (m, 1H), 3.58 (s, 3H), 3.47-3.42 (m,1H), 3.29 (dd, J=3.3, 10.1 Hz, 1H), 2.68 (m, 2H), 2.56-2.43 (m, 2H),2.14 (s, 3H), 2.12 (s, 3H), 2.07-2.04 (m, 2H), 2.00 (s, 3H), 1.17-1.57(m, 2H), 0.83 (s, 9H), −0.01 (s, 3H), −0.06 (s, 3H); ¹³C-NMR (125 MHz,CDCl₃) δ 206.2, 171.4, 170.9, 170.4, 170.1, 168.9, 138.1, 138.2, 138.0,137.8, 129.2, 128.6, 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7,115.1, 101.2, 98.0, 97.3, 82.5, 77.9, 77.4, 77.2, 76.2, 75.1, 74.5,73.9, 73.5, 73.2, 71.7, 69.8, 69.4, 62.9, 61.7, 52.8, 51.8, 37.9, 30.0,28.7, 28.0, 25.7, 21.1, 21.0, 27.9, −4.6, −5.1; FAB MS (C₆₁H₈₁N₃O₂₁Si)m/z (M)⁺ calcd 1219.5132, obsd 1219.5107.

n-Pentenyl(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosiduronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate 75

Compound 74 (45 mg, 0.037 mmol) was dissolved in THF (5 mL). Glacialacetic acid (1.3 mL) and HF/pyridine-complex (0.8 mL) were added and thesolution was stirred at room temperature for three days. The mixture waspoured into Et₂O and washed with brine, water, sat. NaHCO₃ and driedover Na₂SO₄. After filtration, the solvent was removed under reducedpressure. Flash chromatography on silica gel (hexanes/EtOAc 7:3)afforded 75 (33 mg, 0.03 mmol, 82%) as a colorless foam. ¹H-NMR (500MHz, CDCl₃) δ 7.39-7.27 (m, 15H), 5.49 (d, J=3.9 Hz, 1H), 5.07-4.95 (m,4H), 4.90 (bs, 1H), 4.85 (bs, 1H), 4.83-4.73 (m, 4H), 4.65-4.62 (m, 4H),4.47 (d, J=7.0 Hz, 1H), 4.35 (dd, 1H), 4.24-4.19 (m, 2H), 3.99 (d, J=9.0Hz, 2H), 3.96 (m, 1H), 3.87-3.81 (m, 3H), 3.74-3.71 (m, 2H), 3.66 (s,3H), 3.58 (dd, 1H), 3.48 (s, 3H), 3.48-3.43 (m, 1H), 3.28 (dd, J=3.7,10.4 Hz, 1H), 2.68 (t, 2H), 2.60-2.45 (m, 3H), 2.14-2.07 (m, 11H),1.68-1.60 (m, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ 206.2, 171.4, 170.8,169.7, 169.3, 168.8, 138.1, 137.8, 137.8, 137.3, 129.2, 128.8, 128.6,128.4, 128.3, 127.9, 127.7, 127.7, 115.1, 101.2, 98.3, 97.6, 82.5, 78.4,77.4, 75.1, 75.1, 74.8, 74.7, 74.6, 74.4, 73.6, 72.9, 69.8, 69.5, 69.0,67.9, 67.7, 63.5, 61.9, 52.8, 52.3, 37.9, 30.0, 29.9, 28.7, 28.0, 21.6,21.1; FAB MS (C₅₅H₆₇N₃O₂₁) m/z (M)⁺ calcd 1105.4267, obsd 1105.4252.

tert-Butyldimethylsilyl(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosyl)-(1→4)-3,6-di-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside76

Compound 55 (365 mg, 0.38 mmol) and 7 (105 mg, 0.26 mmol) werecoevaporated with toluene and dried under vacuum for 1 h. The mixturewas dissolved in CH₂Cl₂ (3 mL) and after cooling to −25° C., TMSOTf (20μL, 0.1 M in CH₂Cl₂) was added. The mixture was stirred for 4 h and thendiluted with CH₂Cl₂ and filtered through a pad of Celite. The solventwas removed under reduced pressure and the residue was purified bysilica gel column chromatography (toluene:EtOAc 95:5→80:20) to yield 76(195 mg, 0.16 mmol, 63%) as a syrup. ¹H-NMR (500 MHz, CDCl₃) δ 7.37-7.24(m, 15H), 5.40 (d, J=3.7 Hz, 1H), 4.99 (t, 1H), 4.92-4.87 (m, 3H),4.84-4.70 (m, 3H), 4.67 (d, J=10.6 Hz, 1H), 4.60 (d, J=7.6 Hz, 1H), 4.57(d, J=10.9 Hz, 1H), 4.50-4.47 (d, J=10.4 Hz, 1H), 4.42 (d, J=7.6 Hz,1H), 4.27-4.12 (m, 4H), 3.90 (d, J=9.5 Hz, 1H), 3.85 (dd, J=8.5, 10.4Hz, 1H), 3.78-375 (m, 1H), 3.76 (s, 1H), 3.73-3.60 (m, 2H), 3.55-3.50(m, 2H), 3.32-3.27 (m, 2H), 2.74-2.63 (m, 2H), 2.59-2.54 (m, 2H), 2.12(s, 3H), 2.11 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 0.9 (s 9H), 0.15 (s,3H), 0.14 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 206.1, 171.7, 170.8,170.7, 169.8, 168.1, 145.7, 141.5, 137.7, 137.7, 137.6, 128.7, 128.7,128.6, 128.6, 128.3, 128.3, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5,101.3, 97.6, 97.6, 82.5, 80.3, 76.8, 75.7, 75.2, 74.8, 74.7, 74.6, 73.1,72.8, 71.8, 69.9, 66.5, 63.4, 62.5, 62.3, 52.9, 37.9, 37.7, 29.9, 27.8,25.7, 21.1, 21.0, 20.9, 18.2, −4.3, −5.0; ES MS (C₅₇H₇₄N₆O₂₀Si) m/z (M)⁺calcd 1190.4727, obsd 1190.4735.

O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosyl)-(1→4)-(3,6-di-O-acetyl-2-azido-2-deoxy-α/β-D-glucopyranosyl)trichloroacetimidate 77

A solution of 76 (80 mg, 0.07 mmol) in THF (1 mL) was cooled to 0° C.Glacial acetic acid (10 μL, 0.17 mmol) and TBAF (1M in THF, 110 μL, 0.11mmol) were added in sequence. After 30 min the mixture was poured intoEt₂O (100 mL) and washed with brine (3×). The organic layer was driedover Na₂SO₄, filtered and the solvent was removed under reducedpressure. The residue was dissolved in CH₂Cl₂ (20 mL) and the solutionwas cooled to 0° C. Trichloroacetonitrile (190 μL, 1.9 mmol) and DBU (5μL, 0.03 mmol) were added and the mixture was stirred at 0° C. for 1 hand at room temperature for 3 h. After concentration, flashchromatography on silica gel (hexanes:EtOAc 85:15→70:30) afforded 77 (82mg, 0.07 mmol, 87%) as a colorless foam. FAB MS (C₅₃H₆₀Cl₃N₇O₂₀) m/z(M)⁺ calcd 1219.2959, obsd 1219.2963.

n-Pentenyl(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosyl)-(1→4)-(3,6-di-O-acetyl-2-azido-2-deoxy-α/β-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosiduronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate 78

Compound 75 (50 mg, 0.04 mmol) and 77 (30 mg, 0.03 mmol) werecoevaporated with toluene and dried under vacuum for 1 h. The mixturewas dissolved in toluene (1 mL) and after cooling to −25° C., TBSOTf (20μL, 0.1M in CH₂Cl₂) was added. The mixture was stirred for 2 h and thendiluted with CH₂Cl₂ and filtered through a pad of Celite. The solventwas removed under reduced pressure and the residue was purified bysilica gel column chromatography (toluene:EtOAc 95:5→80:20) to yield 78(36 mg, 0.02 mmol, 62%) as a syrup. [α]²⁴ _(D): +35 (c 0.70, CH₂Cl₂);¹H-NMR (500 MHz, CDCl₃) δ 7.36-7.17 (m, 30H), 5.77-5.83 (m, 1H),5.48-5.45 (m, 2H), 5.41 (d, J=3.7 Hz, 1H), 5.33 (t, 1H), 5.29 (d, J=3.7Hz, 1H), 5.17-4.90 (m, 7H), 4.87-4.80 (m, 6H), 4.78-4.61 (m, 7H),4.57-4.53 (m, 2H), 4.45 (d, J=7.9 Hz, 1H), 4.39 (d, J=7.9 Hz, 1H),4.38-4.22 (m, 2H), 4.20-4.10 (m, 2H), 3.96 (d, J=9.4 Hz, 1H), 3.95-3.84(m, 2H), 3.82-3.66 (m, 6H), 3.75 (s, 3H), 3.71 (s, 3H), 3.67 (s, 3H),3.54-3.49 (m, 3H), 3.48-3.43 (m, 1H), 3.30 (dd, J=3.9, 10.4, Hz, 1H),3.16 (dd, J=3.3, 10.1, Hz, 1H), 3.11 (dd, J=3.0, 10.4, Hz, 1H),2.69-2.63 (m, 4H), 2.58-2.48 (m, 4H), 2.16 (s, 3H), 2.14 (s, 3H), 2.12(s, 3H), 2.04 (s, 3H), 2.09-2.02 (m, 2H), 2.02 (s, 3H), 1.87 (s, 3H),1.66-1.60 (m, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ 207.2, 206.2, 171.4,171.2, 171.0, 170.8, 170.7, 170.6, 170.1, 170.0, 169.0, 168.1, 138.2,137.8, 137.7, 137.6, 129.2, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3,128.1, 127.9, 127.8, 127.5, 127.3, 125.5, 115.2, 101.5, 101.2, 99.6,98.1, 97.7, 97.4, 82.9, 82.6, 80.4, 78.0, 77.4, 76.9, 75.8, 75.6, 75.2,74.9, 74.8, 74.6, 74.4, 74.3, 73.6, 73.1, 71.5, 71.0, 70.0, 69.5, 69.3,63.5, 63.3, 62.3, 61.9, 61.7, 61.0, 52.9, 52.8, 52.5, 37.9, 37.5, 31.2,30.1, 30.0, 29.2, 28.7, 28.1, 27.7, 21.7, 21.2, 21.0, 20.9, 20.8; ES MS(C₁₀₆H₁₂₅N₉O₄₀) m/z (M+Na)⁺ calcd 2186.7916, obsd 2186.7984.

n-Pentenyl(3,6-di-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-3-O-benzyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl2,3-di-O-benzyl-β-D-glucopyranosiduronate 79

A solution of 70 (93 mg, 0.07 mmol) and thiourea (230 mg, 3.02 mmol) inDMF/pyridine (10/1, 2 mL) was stirred for 24 h. After removal of thesolvent under reduced pressure, the residue was dissolved in CHCl₃ andfiltered. The solvent was removed under reduced pressure and flashchromatography on silica gel affordedpent-4-enyl(3,6-di-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-3-O-benzyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate (79 mg, 0.06 mmol, 90%). [α]²³_(D): +12.5 (c 0.80, CHCl₃); IR (thin film on NaCl) 2914, 2364, 2108,1743, 1371; ¹H-NMR (500 MHz, CDCl₃) δ 7.38-7.28 (m, 15H arom.),5.85-5.77 (m, 1H, CH olef.), 5.57 (d, J=3.5 Hz, 1H, H-1C), 5.29 (d,J=4.5 Hz, 1H, H-1B), 5.22 (dd, J=10.5, 9.0 Hz, 1H, H-3A), 5.07 (d, J=3.0Hz, 1H, H-1A), 5.02-4.97 (m, 3H, CH₂ olef., 1H of CH ₂Ph), 4.93-4.90 (m,2H, H-2B, 1H of CH ₂Ph), 4.82-4.80 (A part of AB system, J_(AB)=11.0 Hz,1H of CH ₂Ph), 4.76-4.68 (m, 3H of CH ₂Ph), 4.63 (d, J=4.5 Hz, 1H,H-5B), 4.49 (A part of ABX system, J=12.5, 3.5 Hz, 1H, H-6_(a)), 4.31(d, J=7.5 Hz, 1H, H-1D), 4.29-4.24 (m, 2H, H-6_(a), H-⁶ _(b)), 4.16 (Bpart of ABX system, J=12.5, 2.0 Hz, 1H, H-6_(b)), 4.09-4.05 (m, 2H,H-4B, 1H), 4.00-3.84 (m, 5H, H-5A, H-4C, 1H of OCH₂, 3H), 3.77-3.71 (m,2H, H-3D, H-3C), 3.67 (s, 3H, COOMe), 3.66 (s, 3H, COOMe), 3.62(dt,J=7.5, 2.0 Hz, 1H, H-2D), 3.54-3.50 (m, 2H, 1H of OCH₂, 1H), 3.44(dt,J=9.5, 5.0 Hz, 1H, H-4A), 3.27 (dd, J=10.5, 4.0 Hz, 1H, H-2C), 3.17 (dd,J=10.5, 3.5 Hz, 1H, H-2A), 3.03 (d, J=5.0 Hz, 1H, OH-4A), 2.34 (d, J=2.0Hz, 1H, OH-2D), 2.20-2.05 (m, 14H, 4 CH₃, pent-CH₂), 1.77-1.68(m, 2H,pent-CH₂); ¹³C-NMR (125 MHz, CDCl₃) δ 172.0, 171.3, 171.1, 170.3, 169.8,169.0, 138.4, 138.2, 138.1, 137.4, 128.7, 128.6, 128.4, 128.3, 128.2,128.0, 127.9, 127.8, 115.3, 103.1, 98.6, 98.4, 97.5, 83.9, 78.2, 75.8,75.0, 74.9, 74.8, 74.7, 74.0, 73.9, 72.4, 71.4, 70.1, 70.0, 69.9, 69.6,69.0, 63.3, 62.6, 62.0, 61.1, 52.8, 52.4, 30.3, 28.8, 21.1, 21.0, 20.9;ES MS (C₆₀H₇₄N₆O₂₅) m/z (M+Na)⁺ calcd 1301.4601, obsd 1301.4596.

A solution ofn-pentenyl(3,6-di-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-3-O-benzyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate (62 mg, 0.05 mmol) in CH₂Cl₂ (2.0mL) was added to freshly activated powdered 4 Å molecular sieves (60mg). Benzyl bromide (29 μL, 0.24 mmol) was added and the mixture wasstirred at room temperature. After 30 minutes, Ag₂O (67 mg, 0.29 mmol)was added and the mixture was stirred overnight. The mixture wasfiltered and the solvent was removed under reduced pressure and flashchromatography on silica gel afforded 79 (50 mg, 0.04 mmol, 76%) as apale yellow oil. [α]²⁴ _(D): +23.9 (c 1.80, CHCl₃); IR (thin film onNaCl) 2924, 2108, 1743, 1496, 1453; ¹H-NMR (500 MHz, CDCl₃) δ 7.36-7.25(m, 20H arom.), 5.84-5.76 (m, 1H, CH olef.), 5.54 (d, J=4.0 Hz, 1H,H-1C), 5.28 (d, J=4.0 Hz, 1H, H-1B), 5.21 (t, J=9.5 Hz, 1H, H-3A), 5.07(d, J=3.5 Hz, 1H, H-1A), 5.03-4.90 (m, 6H, CH₂ olef., H-2B, 3H of CH₂Ph), 4.82-4.67 (m, 5H of CH ₂Ph), 4.62 (d, J=4.0 Hz, 1H, H-5B),4.50-4.45 (m, 2H, H-6_(a)A, H-1D), 4.31-4.29 (A part of ABX system,J=12.0, 1.0 Hz, H-6_(a)C), 4.26-4.23 (B part of ABX system, J=12.0, 3.5Hz, H-6_(b)C), 4.17-4.14 (B part of ABX system, J=12.5, 2.5 Hz,H-6_(b)A), 4.09-4.05 (m, 2H, H-4B, H-4D), 3.96-3.89 (m, 4H, H-5A, H-3B,H-5D, 1H of OCH₂), 3.86 (t, J=9.5 Hz, 1H, H-4C), 3.76-3.73 (m, 2H, H-3C,H-3D), 3.67 (s, 3H, COOCH₃), 3.65 (s, 3H, COOCH₃), 3.57-3.49 (m, 3H,H-5C, H-2D, 1H of OCH₂), 3.44 (td, J=10.5, 3.5 Hz, 1H, H-2A), 3.24 (dd,J=4.0, 10.0 Hz, 1H, H-2C), 3.17 (dd, J=3.5, 10.5 Hz, 1H, H-2A), 3.02 (d,J=5.0 Hz, 1H, OH), 2.16-2.04 (m, 14H, 3 CH₃, pent-CH₂), 1.77-1.70 (m,2H, pent-CH₂); ¹³C-NMR (125 MHz, CDCl₃) δ 172.0, 171.3, 171.1, 170.3,169.7, 169.1, 138.3, 138.2, 138.1, 137.4, 128.7, 128.6, 128.5, 128.4,128.3, 128.2, 128.0, 127.8, 127.7, 115.3, 103.9, 98.6, 98.4, 97.4, 84.1,81.9, 78.1, 75.75, 75.74, 75.4, 75.0, 74.9, 74.8, 74.4, 74.0, 73.8,72.4, 71.4, 70.1, 70.0, 69.9, 69.5, 68.9, 63.2, 62.6, 62.0, 61.1, 52.8,52.4, 30.3, 29.0, 21.0, 21.1, 20.9; ES MS (C₆₇H₈₀N₆O₂₅) m/z (M+Na)⁺calcd 1391.5070, obsd 1391.5107.

n-Pentenyl(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate 80

A solution ofpent-4-enyl(3,6-di-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-3-O-benzyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-β-D-glucopyranosiduronate (50 mg, 0.04 mmol), pyridine (6.3μL, 0.08 mmol), acetic anhydride (8, μL, 0.08 mmol) and catalytic DMAPwas stirred at room temperature for 6 h. After removal of the solventunder reduced pressure, flash chromatography on silica gel(hexanes:EtOAc 1.5:1) affordedn-pentenyl(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate (57 mg, 0.08 mmol,quantitative) as a yellow oil. [α]²³ _(D): +165 (c 0.10, CHCl₃); ¹H-NMR(500 MHz, CDCl₃) δ 7.38-7.24 (m, 15H arom.), 5.82-5.74 (m, 1H, CHolef.), 5.48 (d, J=4.0 Hz, 1H, H-1A), 5.34 (t, J=9.5 Hz, 1H, H-3A), 5.27(d, J=4.0 Hz, 1H, H-1B), 5.08 (d, J=4.0 Hz, 1H, H-1C), 5.06-4.88 (m, 6H,H-2D, H-4A, CH₂ olef, H-2B, 1H of CH ₂Ph), 4.79-4.66 (m, 6H, 5H of CH₂Ph, H-5B), 4.47 (d, J=7.5 Hz, 1H, H-1D), 4.32-4.23 (m, 2H, H-6_(a)C,H-6_(b)C), 4.22-4.18 (m, 2H, H-4D, 1H), 4.14-4.04 (m, 3H, H-4B, 2H),3.99 (d, J=9.5 Hz, 1H, H-5D), 3.94 (t, J=50 Hz, 1H, H-3B), 3.87-3.82 (m,3H, H-4C, 2H), 3.74 (t, J=10.0 Hz, 1H, H-3C), 3.67 (s, 3H, COOCH₃), 3.64(s, 3H, COOCH₃), 3.57-3.55 (m, 1H, H-5C), 3.47-3.43 (m, 1H), 3.29-3.26(m, 2H, H-2A, H-2C), 2.68 (t, J=6.5 Hz, 2H,lev-CH₂), 2.57-2.44 (m,2H,lev-CH₂), 2.14-2.01 (m, 20H, 6 CH₃, pent-CH₂), 1.72-1.59 (m, 2H,pent-CH₂); ¹³C-NMR (125 MHz, CDCl₃) δ 206.2, 171.4, 171.0, 170.7, 170.2,170.1, 169.7, 169.6, 168.8, 138.1, 138.0, 137.8, 137.3, 128.7, 128.6,128.4, 128.3, 128.2, 127.9, 127.8, 115.1, 101.2, 98.4, 98.3, 97.4, 82.5,78.2, 75.6, 75.4, 75.0, 74.5, 74.4, 73.9, 73.6, 70.3, 69.8, 69.6, 69.4,68.6, 68.2, 63.3, 61.9, 61.5, 61.1, 52.8, 52.4, 37.9, 30.0, 28.7, 28.0,21.0, 20.9, 20.87, 20.83, 20.7; ES MS (C₆₇H₈₂N₆O₂₈) m/z (M+Na)⁺ calcd1441.5069, obsd 1441.5098.

A solution ofn-pentenyl(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl2-O-acetyl-3-O-benzyl-α-_(L)-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-methyl3-O-benzyl-2-O-levulinyl-β-D-glucopyranosiduronate (42 mg, 0.03 mmol) inpyridine/AcOH (3/2 0.3 mL) was cooled to 0° C. and hydrazine hydrate(7.4 mg, 0.15 mmol) was added. After 20 minutes acetone 82 mL) was addedand the ice bath was removed. After stirring the mixture at roomtemperature for 30 minutes, the solvent was removed under reducedpressure and the residue was purified by silica gel columnchromatography (hexanes:EtOAc 1:1) to afford 80 (35 mg, 0.03 mmol, 90%)as a pale yellow oil. [α]²³ _(D): +138 (c 3.6, CHCl₃); ¹H-NMR (500 MHz,CDCl₃) δ 7.38-7.26 (m, 15H arom.), 5.84-5.76 (m, 1H, CH olef.), 5.58 (d,J=3.5 Hz, 1H, H-1A), 5.34 (dd, J=10.5, 9.5 Hz, 1H, H-3A), 5.27 (d, J=4.0Hz, 1H, H-1B), 5.08 (d, J=3.5 Hz, 1H, H-1C), 5.05-4.97 (m, 4H, CH₂olef., H-4A, 1H), 4.92 (t, J=4.5 Hz, 1H, H-2B), 4.89-4.87 (A part of ABsystem, J_(AB)=10.5 Hz, 1H of CH ₂Ph), 4.82-4.79 (B part of AB system,J_(AB)=10.5 Hz, 1H of CH ₂Ph), 4.76-4.67 (m, 4H), 4.32-4.19 (m, 4H),4.11-4.04 (m, 4H), 3.97-3.90 (m, 3H), 3.85 (t, J=9.5 Hz, 1H), 3.77-3.71(m, 2H), 3.67-3.60 (m, 7H, 2 COOMe, 1H), 3.55-3.49 (m, 2H), 3.29-3.25(m, 2H, H-2C, H-2A), 2.40 (bs, 1H, OH), 2.14-2.02 (m, 17H, 5 CH₃,Pent-CH₂), 1.76-1.69 (m, 2H, pent-CH₂); ¹³C-NMR (500 MHz, CDCl₃) δ171.0, 170.7, 170.3, 170.1, 169.8, 169.6, 168.9, 138.3, 138.1, 138.0,137.3, 128.7, 128.6, 128.4, 128.3, 128.2, 127.9, 127.8, 115.3, 103.1,98.4, 97.5, 83.9, 78.2, 75.6, 75.3, 74.9, 74.8, 74.7, 73.9, 70.3, 70.0,69.5, 69.3, 68.6, 68.2, 63.3, 61.9, 61.4, 61.0, 52.8, 52.4, 30.3, 28.7,21.1, 20.90, 20.88, 20.86, 20.79. ES MS (C₆₂H₇₆N₆O₂₆) m/z (M+Na)⁺ calcd1338.5152, obsd 1338.4932.

n-Pentenyl(2-deoxy-2-sodium sulfonatamido-3,4,6-tri-O-sodiumsulfonato-α-D-glucopyranosyl)-(1→4)-(sodium 2-O-sodiumsulfonato-α-D-idopyranosyluronate)-(1→4)-(2-deoxy-2-sodiumsulfonatamido-6-O-sodium sulfonato-α-D-glucopyranosyl)-(1→4)-sodium2-O-sodium sulfonato-β-D-glucopyranosiduronate 81

A solution of 80 (44 mg, 0.03 mmol) in THF (3.0 mL) was cooled to −13°C. and 50% H₂O₂ (1.0 mL) and 0.7 M aq LiOH (0.8 mL) were added dropwise.The mixture was warmed to 0° C. over one hour and at room temperatureovernight. Sodium hydroxide solution (4 M, 0.8 mL) was added and themixture was stirred overnight. After acidification to pH 6.0 with 3 MHCl in MeOH, the solvent was partially removed under vacuum. Thesolution was diluted with EtOAc and the two phases were separated. Theorganic phase was washed twice with acidified aqueous sulfite (pH 3.5)and dried over Na₂SO₄. After filtration, the solvent was removed underreduced pressure and the residue was purified on Sephadex LH20(CH₂Cl₂:EtOH 1:1) affording n-pentenyl(2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-benzyl-α-L-idopyranosyluronicacid)-(1→4)-(2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-benzyl-β-D-glucopyranosiluronicacid (27 mg, mmol, 82%) as a colorless oil. ES MS (C₅₀H₆₂N₆O₂₁) m/z(M+Na)⁺ calcd 1105.3865, obsd 1105.3806.

A solution ofn-pentenyl(2-azido-2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-benzyl-α-L-idopyranosyluronicacid)-(1→4)-(2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-benzyl-β-D-glucopyranosiluronicacid (35 mg, 0.03 mmol) and sulfur trioxide triethylamine complex (88mg, 0.48 mmol) in DMF (1.5 mmol) was stirred under nitrogen at 50° C.for 20 h. Aqueous NaHCO₃ (10%, 3 mL) was added at room temperature andthe mixture was stirred for 3.5 h. The reaction mixture wasconcentrated, dissolved in MeOH and filtered through a pad of Celite.After removal of the solvent under reduced pressure, the residue waspurified through a Sephadex G-25 column eluted with 0.2 N NaCl. Afterconcentration and desalting through a Sephadex G-25 column eluted withwater, n-pentenyl(2-azido-2-deoxy-3,4,6-tri-O-sodium sulfonato-α-Dglucopyranosyl)-(1→4)-(sodium 3-O-benzyl-2-O-sodiumsulfonato-α-D-idopyranosyluronate)-(1→4)-(2-azido-3-O-benzyl-2-deoxy-6-O-sodiumsulfonato-α-D-glucopyranosyl)-(1→4)-sodium 3-O-benzyl-2-O-sodiumsulfonato-β-D-glucopyranosiduronate (25 mg, 0.015 mmol, 50%) wasobtained as a colorless solid. ¹H-NMR (500 MHz, D₂O) δ 7.39-7.15 (m, 15Harom.), 5.82-5.74 (m, 1H, CH olef.), 5.25 (d, J=4.0 Hz, 1H, H-1C), 5.17(bs, 1H, H-1B), 5.07 (d, J=3.5 Hz, 1H, H-1A), 4.99-4.87 (m, 2H, CH₂olef.), 4.72-4.66 (m, 7H, 3 CH ₂Ph, H-1D), 4.42-4.38 (m, 2H), 4.32-4.28(m, 2H), 4.20-4.10 (m, 7H), 3.96-3.69 (m, 8H), 3.55-3.50 (m, 1H), 3.43(dd, J=10.5, 3.5 Hz, 1H, H-2A), 3.34 (dd, J=10.0, 4.0 Hz, 1H, H-2C),2.06-2.02 (m, 2H, pent-CH₂), 1.61-1.55 (m, 2H, pent-CH₂); ¹³C-NMR (125MHz, D₂O) δ139.7, 137.9, 137.4, 137.3, 129.9, 129.4, 129.2, 129.2,128.9, 128.8, 115.4, 101.4, 98.0, 93.6, 82.8, 80.6, 78.6, 77.3, 76.5,76.0, 75.3, 75.6, 75.3, 74.6, 73.0, 72.7, 71.4, 70.7, 70.4, 69.3, 68.6,68.4, 67.4, 66.9, 63.6, 62.2, 29.9, 28.6; ES MS (C₅₀H₆₂N₆O₃₉S₆) m/z(M−2H)²⁻ calcd. 780.0605, obsd. 780.0564.

A solution of n-pentenyl(2-azido-2-deoxy-3,4,6-tri-O-sodiumsulfonato-α-Dglucopyranosyl)-(1→4)-(sodium 3-O-benzyl-2-O-sodiumsulfonato-α-D-idopyranosyluronate)-(1→4)-(2-azido-3-O-benzyl-2-deoxy-6-O-sodiumsulfonato-α-D-glucopyranosyl)-(1→4)-sodium 3-O-benzyl-2-O-sodiumsulfonato-β-D-glucopyranosiduronate (25 mg, 0.02 mmol) in EtOH/water(2/1, 6.0 mL) was treated by a stream of hydrogen in the presence ofPd/C catalyst (10%, 40 mg) for three days. After filtration on a pad ofCelite, the solvent was removed under reduced pressure. ¹H-NMR (500 MHz,D₂O) δ 5.41 (bs, 1H), 5.29 (bs, 1H), 5.10 (bs, 1H), 4.78 (bs, 1H),4.60-4.28 (m, 2H), 4.24-3.57 (m, 17H), 3.55-3.41 (m, 2H), 3.13 (bs, 1H),1.46-1.41 (m, 2H), 1.18-1.12 (m, 4H), 0.70 (t, J=7.0 Hz, 3H); ES MS(C₂₉H₅₀N₂O₃₉S₆) m/z (M−2H)²⁻ calcd. 620.0085, obsd 620.0040.

The residue (20 mg, 0.02 mmol) was dissolved in water (4 mL). Sulfurtrioxide pyridine complex (101 mg, 0.6 mmol) was added in five portionsevery 30 minutes with the pH being maintained at 9.5 by addition of 4NNaOH. After 3.5 h, the reaction mixture was concentrated and purifiedthrough a Sephadex G-25 column eluted with 0.2 N NaCl. Afterconcentration and desalting through a Sephadex G-25 eluted with water,81 (13 mg, 0.01 mmol, 60%) was obtained as a solid. [α]²³ _(D): +57 (c1.2, CHCl₃); ¹H-NMR (500 MHz, D₂O) δ 5.47 (d, J=3.0 Hz, 1H), 5.42 (d,J=3.0 Hz, 1H), 5.03 (bs, 1H), 4.48 (d, J=8.0 Hz, 1H), 4.37-4.00 (m, 9H),3.97 (t, J=8.0 Hz, 1H), 3.86-3.81 (m, 2H), 3.76-3.68 (m, 4H), 3.54-3.51(m, 2H), 3.46 (dd, J=10.5, 3.0 Hz, 1H), 3.14 (dd, J=10.0, 3.5 Hz, 1H),1.50-1.45 (m, 2H), 1.22-1.16 (m, 6H), 0.74 (at, J=7.0 Hz, 3H); HSQCanomeric cross peaks (D₂O) δ (4.48×100.8), (5.03×99.8), (5.42×98.2),(5.47×95.4). ES MS (C₂₉H₄₄N₂O₄₅S₈Na₆) m/z (M)⁺ calcd. 1533.8, obsd.1534.1.

INCORPORATION BY REFERENCE

All of the patents and publications cited herein are hereby incorporatedby reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-5. (canceled)
 6. A trisaccharide selected from the group consistingof:

wherein X represents independently for each occurrence hydroxyl,acyloxy, silyloxy, halide, alkylthio, arylthio, alkoxy, aryloxy, or—OC(NH)CCl₃; R represents independently for each occurrence H, alkyl,aryl, arylalkyl, heteroarylalkyl, silyl, acyl, alkenyloxycarbonyl, oraralkyloxycarbonyl; and R′ represents independently for each occurrenceH, alkyl, aryl, arylalkyl, or heteroarylalkyl.
 7. The trisaccharide ofclaim 6, wherein X represents fluoro, bromo, 4-pentenyloxy or—OC(NH)CCl₃.
 8. The trisaccharide of claim 6, wherein R′ representsindependently for each occurrence alkyl.
 9. The trisaccharide of claim6, wherein X represents fluoro, bromo, 4-pentenyloxy or —OC(NE)CCl₃; andR′ represents independently for each occurrence alkyl.
 10. Thetrisaccharide of claim 6, wherein said trisaccharide is selected fromthe group consisting of:

wherein X is silyloxy or —OC(NH)CCl₃; and R is H or silyloxy.
 11. Amethod of preparing a glycosaminoglycan, comprising the step of:reacting a first mono-, di- or tri-saccharide, comprising an activatedanomeric carbon, with a second mono-, di- or tri-saccharide, comprisinga hydroxyl or amino group, to form an oligosaccharide, comprising aglycosidic linkage between said anomeric carbon of said first mono-, di-or tri-saccharide and said hydroxyl or amino group of said second mono-,di- or tri-saccharide.
 12. The method of claim 11, wherein the firstmono-, di- or tri-saccharide is not identical to the second mono-, di-or tri-saccharide.
 13. The method of claim 11, wherein neither the firstmono-, di- or tri-saccharide nor the second mono-, di- or tri-saccharideis covalently linked to a solid support.
 14. The method of claim 11,wherein the first first mono-, di- or tri-saccharide or the secondmono-, di- or tri-saccharide is covalently linked to a solid support.15. The method of claim 14, further comprising the step of: cleavingsaid covalent linkage between said oligosaccharide and said solidsupport with an alkene metathesis catalyst and an alkene.
 16. The methodof claim 1 1, further comprising the step of: sulfating a hydroxyl oramino moiety of said oligosaccharide.
 17. The method of claim 11,further comprising the step of: removing a hydroxyl or amino protectinggroup from said oligosaccharide by hydrogenolysis. 18-22. (canceled)